Replace with: A MAN

replace with: A MAN

This isn't in Crangle's edition. if we follow it, this should be deleted

replace with: continuing

replace with: to

italics

replace with: DIANA

replace with: By

replace with: holding

no line break before: tomorrow...

add line break before (turning the first three dots should be next to eyes, not in a new line

replace with: left-hand

replace with: Pass

add line break after full stop

replace hyphen with em-dash

add line break before: No use

delete: up

add line break before Those

no line break between first and dots

replace with: shoulders

replace hyphens with em-dashes

no line break after mud

line break after you....?

replace hyphen with em-dash

replace with: anxiously

replace with: LOONY

replace with: DIANA

the three dots should be next to know

replace hyphen with em-dash

replace with: DIANA

delete: he

line break after the bracket and three dots after pig

replace with: snoozily

replace with: DIANA

replace with: LOONY

replace with: PICKED PEOPLE

replace with: LOONY

indented in Crangle

replace with: DIANA

I would replace "he" with "Loony" to make the sentence clearer and more elegant

replace with: DIANA

replace with: bric-à-brac line should be indented (as in Crangle)

indented in Crangle

no line break between round and numbers

replace with: And

replace with: talk about something!

replace with: warm-footed

replace with: DIANA delete full stop

replace with: to

replace with: back) - the latter is an em-dash

replace with: DIANA's

line break after dots

delete full stop

the dots should be next to submerged

delete endnote

delete endnote

replace with: LOONY's

replace with dash

replace with: DIANA

the dots should be next to caress, not in a new line

Could we add a refence to my book here, where I write about this issue? Also, if we want to keep our initials for the notes, this would end with: (LS). The reference to my book is: See L. Scuriatti, Mina Loy's Critical Modernism (Gainesville: University of Florida Press, 2019), pp. 113-116.

add space. It should read: museums and

replace "souls to" with: soul's

replace with: china

replace "his" with: the

replace with: anticipate

replace "for autumn" with: for the autumn

replace with: "Has to have"

replace with: DIANA

replace with: Futurism

Replace with HOUSELESS LOONY

replace with LADY DIANA

no line break

italics

There does not seem to be a line break in Crangle's edition, but it is not clear. Is there a break in The Dial?

delete full stop

replace with FRIENDS

dots should be next to "you", and not in a new line

the three dots should not be on a new line, but next to frost

it should read "into the Arabic" - although it sounds wrong. Either we correct it and add [sic], or insert an excision sign to show that we have intervened onto the text

delete full stop

Italics. "With" should be "with"

italics

italics

delete "his"

delete commas. It should read: Ossy you know

delete comma

insert space between note and knows

hyphenate: steam-heating

delete is

italics

italics

Capitalized: People

Résumé de la vidéo [00:00:03][^1^][1] - [00:03:49][^2^][2]:

Cette vidéo présente un entretien pour un poste de directeur des opérations, qui s'avère être le rôle d'une mère. Les candidats réagissent à la description du poste, qui exige une disponibilité constante, aucune pause, et une multitude de compétences, sans salaire. La révélation que des milliards de personnes, les mères, occupent déjà ce poste, suscite admiration et gratitude.

Points forts: + [00:00:24][^3^][3] Description du poste * Intitulé : Directeur des opérations * Exige une grande mobilité et endurance * Travail debout et activité constante + [00:01:02][^4^][4] Horaires exigeants * 135 heures par semaine, potentiellement 24/7 * Pas de pauses, même pour manger * Nécessite d'excellentes compétences en négociation et relationnelles + [00:01:57][^5^][5] Sacrifices personnels * Pas de vie personnelle, pas de vacances * Charge de travail accrue pendant les fêtes * Attitude positive exigée en tout temps + [00:02:34][^6^][6] Aucune rémunération * Le poste ne prévoit aucun salaire * Réaction de surprise et d'incrédulité des candidats * Révélation que le poste est celui d'une mère

Résumé de la vidéo [00:02:40][^1^][1] - [00:03:49][^2^][2]:

La vidéo présente un entretien pour un poste de directeur des opérations, qui s'avère être une métaphore pour le rôle d'une mère. Les candidats sont choqués par les exigences extrêmes du poste, qui incluent une disponibilité 24/7, aucune pause, et des compétences dans divers domaines, le tout sans salaire. La révélation que des milliards de personnes, les mères, occupent déjà ce poste, suscite admiration et gratitude.

Points forts: + [00:02:40][^3^][3] Les exigences du poste * Disponibilité constante, sans pauses * Compétences en médecine, finance, et arts culinaires * Capacité à travailler dans un environnement chaotique + [00:03:01][^4^][4] La révélation sur le poste * Le poste est une métaphore pour le rôle de mère * Les mères rencontrent toutes les exigences sans salaire * Éveil de la reconnaissance pour le travail des mères + [00:03:15][^5^][5] Réactions émotionnelles * Les candidats expriment leur admiration pour les mères * Réflexion sur l'appréciation de leurs propres mères * Les mères sont célébrées pour leur dévouement inconditionnel

eLife assessment

This work identifies the molecular function of an orphan human transporter, SLC35G1, providing convincing but somewhat incomplete evidence that this protein is involved in intestinal citrate absorption. This work provides important insight into transporter function and human physiology.

A policy decision about how often we may have to go to the page allocator.

computes a several values for the allocator based on the size and flags of the allocator being created.

Depending on the CONFIG_SLUB_TINY should ther be an active slab for each CPU?

calculate the order (power of two number of pages) that each slab in this allocator should have.

eLife assessment

This is a valuable study on the diffusion rates of drug molecules in human-derived cells, highlighting that their diffusion behavior depends on their charged state. It proposes that blocking drug protonation enhances diffusion and fractional recovery, suggesting improved intracellular availability of weakly basic drugs. The correlation between pKa and intracellular diffusion is solid and well-supported, but the study would benefit from a more rigorous statistical treatment and a balanced comparison across different types of compounds. Despite these limitations, the findings are significant for drug design and understanding the biophysical behavior of small molecules in cells.

eLife assessment

This useful study draws on published single-cell and spatial transcriptomic data of colon cancer liver metastasis to clarify the pro- and anti-tumorigenic properties of NK cells. The authors discover increased GZMK+ resting NK cells in the tumor tissue and reduced abundance of KIR2DL4+ activated NK cells. However, the evidence is currently incomplete, as the models used to validate the hypothesis and claims are inadequate and lack necessary controls.

Congratulations, Arc motherfuckers with iPads: they finally... pushed the maximize button or whatever.

李沐 按照计算层的数量定义层数

High level membership description

delete

put a testimonial in this container

Change to high level membership description higher up

Differentiator - add container above about why this approach works

layout as columns?

move featured in banner

delete

lay out differently

proven system - banner

if

to what?

in banner

delete

a "magic" formula for computing the amount of memory per zone.

XD this sounds fun

Such is the risk of limiting beliefs.

"He who looks for external validation is not properly grounded in life." -- Marcus Aurelius (20 June 2024 future edit, this must be Epictetus)

In other words, do not care about what others think... Heed their advice, take it into account, but ultimately you must make the decision yourself.

YAML is not generally considered easily editable by humans

DataCite is not a data repository.

doing something that is different from what people consider to be normal or acceptable

doing something that is different from what people consider to be normal or acceptable

AIM for Climate Investments

PREPARE and VACS: $50 million announced at COP28

Agriculture Innovation Mission AIM for Climate

PREPARE and the Vision for Adapted Crops and Soils multi-donor funding platform

delete

and frustrated?

Banner

Pain point in container 1

Hyperlink

Love this!

delete

Plus

You said people join for community Highlight main benefit What's the final impact on the biz: growth?

Target customer pain point Desire Then introduce

nn.CrossEntropyLoss是PyTorch中的一个类，它实现了交叉熵损失函数。交叉熵损失函数常用于多分类问题，它可以度量模型的预测概率分布与真实概率分布之间的差异。

reduction='none'是一个参数，它指定了如何对每个样本的损失进行聚合。'none'表示不进行聚合，即返回一个损失值的向量，向量的每个元素对应一个样本的损失。其他可能的值包括'mean'（返回所有样本损失的平均值）和'sum'（返回所有样本损失的总和）。

在 train_ch3 → train_epoch_ch3 中内置优化器是 l.mean().backwar()

在这个例子中，我们选择'none'是因为我们想要在后续的计算中手动处理每个样本的损失，例如，我们可能想要计算每个样本损失的平均值，或者只关注损失最大的几个样本。

Résumé de la vidéo [00:00:05][^1^][1] - [00:22:49][^2^][2] : La vidéo présente une recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique" menée à l'Université Saint-Louis. Elle aborde les défis de l'échec universitaire et les moyens de le surmonter en réévaluant les méthodes pédagogiques et en soutenant les étudiants de manière plus personnalisée.

Points forts : + [00:00:05][^3^][3] Contexte et objectifs de la recherche * Lancement de la recherche dans le cadre de la fusion des universités * Objectif de comprendre et d'adresser l'échec étudiant * Financement par l'université pour une approche collective + [00:08:01][^4^][4] Méthodologie et résultats préliminaires * Suivi d'une cohorte d'étudiants sur trois ans * Identification de profils d'étudiants et de leurs chances de réussite * Importance de l'adaptation des méthodes pédagogiques aux besoins des étudiants + [00:13:00][^5^][5] Profil des étudiants à l'entrée de l'université * Étudiants investis mais inégalement informés et préparés * Confiance en la capacité de réussir malgré des préparations diverses * Six profils d'étudiants identifiés avec des chances de réussite variables + [00:19:00][^6^][6] Expérience des étudiants à l'université * Difficultés d'adaptation à l'autonomie universitaire * Variabilité dans l'appréciation des méthodes d'enseignement * Nécessité d'ajuster les méthodes de travail pour la réussite

Résumé de la vidéo [00:22:51][^1^][1] - [00:44:26][^2^][2] : La vidéo aborde la recherche-action sur la lutte contre l'échec scolaire et la redéfinition de la relation pédagogique. Elle examine les expériences des étudiants durant le premier quadrimestre à l'université, leurs attentes envers les enseignants et l'institution, et l'impact des profils d'entrée sur la réussite académique.

Points forts : + [00:22:51][^3^][3] Expérience du premier quadrimestre * Transition difficile pour les étudiants * Questions sur les attentes et la manière de répondre à ces attentes * Gestion de l'autonomie et attentes envers les enseignants + [00:23:48][^4^][4] Rôle de l'enseignant * Doit être un expert et proche des étudiants * Importance de l'empathie et de l'accompagnement * Les cours doivent être utiles, structurés et bien soutenus + [00:27:00][^5^][5] Hétérogénéité des expériences étudiantes * Différences dans la façon de vivre l'expérience universitaire * Le profil d'entrée n'influence pas directement l'expérience universitaire * Importance de l'assistance et des ressources institutionnelles + [00:32:01][^6^][6] Facteurs de réussite académique * Objectifs académiques et heures de travail personnel * Impact des profils d'entrée sur la réussite * Nécessité de changer les paramètres de fonctionnement de l'université + [00:37:31][^7^][7] Satisfaction et sens dans l'expérience universitaire * Satisfaction non corrélée à la réussite * Les étudiants les plus satisfaits sont souvent parmi les profils les plus fragiles * Importance de la clarté des attentes et de la communication avec les étudiants + [00:42:07][^8^][8] Recommandations pour l'amélioration * Travailler en amont avec l'enseignement obligatoire * Informer sur l'hétérogénéité des publics * Intégrer des tests réflexifs dans le cursus académique

Résumé de la vidéo [00:44:28][^1^][1] - [01:05:38][^2^][2]:

La vidéo présente une recherche-action sur la lutte contre l'échec scolaire et la redéfinition de la relation pédagogique. Elle aborde les propositions pour améliorer le suivi des étudiants, l'importance de ne pas présumer de leur autonomie, et la nécessité de restructurer le calendrier universitaire.

Points saillants: + [00:44:28][^3^][3] Suivi individualisé des étudiants * Proposition de suivis universels mais personnalisés * Importance d'accompagner les étudiants vers l'autonomie * Nécessité d'évaluer la compréhension des consignes + [00:45:26][^4^][4] Apprentissage du travail personnel * Focalisation sur le travail personnel en dehors des cours * Proposition d'organiser des semaines types pour guider les étudiants * Importance de la quantité et de la qualité du travail personnel + [00:46:24][^5^][5] Réorganisation du cadrimestre * La première session arrive trop tard pour les étudiants * Proposition de découper le premier cadrimestre en deux parties * Importance des retours et feedbacks après les mini-sessions + [00:51:01][^6^][6] Débat politique sur l'éducation * Discussion entre journalistes et politiciens sur les thèmes abordés * Échanges sur l'encadrement des étudiants et l'orientation scolaire * Débat sur l'impact du décret paysage et le calendrier universitaire

Résumé de la vidéo [01:05:39][^1^][1] - [01:23:45][^2^][2]:

La vidéo présente un débat politique sur la recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique". Les intervenants discutent des stratégies pour combattre les inégalités dans l'éducation, notamment en améliorant l'orientation des étudiants et en gérant mieux la transition entre l'enseignement secondaire et supérieur. Ils soulignent l'importance d'une analyse à long terme pour suivre les progrès des étudiants et abordent le rôle des facteurs socio-économiques dans la réussite éducative.

Points saillants: + [01:05:39][^3^][3] Propositions du PS pour l'éducation * Mettre en place un observatoire de la vie étudiante * Améliorer l'orientation et la transition entre les niveaux d'enseignement * Attaquer les déterminants socio-économiques de l'échec + [01:08:27][^4^][4] Approche du PTB sur la taille des classes * Réduire la taille des classes pour une meilleure attention individuelle * Proposer des classes de 15 à 17 élèves jusqu'à 8 ans et environ 20 élèves après * Lier la taille des classes à la diminution des inégalités et à la réussite + [01:12:01][^5^][5] Coût des études universitaires selon le PTB * Réduire les frais pour atténuer les inégalités * Proposer des repas à 2 € et diminuer le prix des côtes * Tendre vers la gratuité du minerval + [01:14:43][^6^][6] Formation des enseignants et assistants * Améliorer la formation des enseignants pour traiter les lacunes du secondaire * Utiliser les compétences des professeurs de promotion sociale * Assurer un suivi adéquat des étudiants pour leur réussite

Résumé de la vidéo [01:23:46][^1^][1] - [01:44:37][^2^][2] : La vidéo présente un débat politique sur la recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique". Les intervenants discutent des moyens d'améliorer l'enseignement supérieur en Belgique, notamment en réformant le financement, en intégrant des tests d'orientation non contraignants et en renforçant l'aide à la réussite dans les cursus universitaires.

Points forts : + [01:23:46][^3^][3] Financement de l'enseignement supérieur * Nécessité de refinancer et d'ouvrir l'enveloppe budgétaire * Financement basé sur le nombre d'étudiants * Importance de la pédagogie dans l'orientation + [01:25:46][^4^][4] Tests d'orientation et aide à la réussite * Tests pour identifier les lacunes des étudiants * Aide à la réussite intégrée au cursus * Premier quadrimestre avec modules pédagogiques transversaux + [01:27:19][^5^][5] Conditions de vie des étudiants * Impact de la précarité étudiante sur la réussite * Propositions pour soutenir les étudiants financièrement * Sortie de l'enveloppe fermée pour réduire la concurrence entre établissements + [01:30:00][^6^][6] Évaluation des acquis de base et contrat d'aide à la réussite * Proposition d'évaluation obligatoire mais non contraignante en juillet * Contrat d'aide à la réussite axé sur la remédiation * Importance de l'orientation et de l'évaluation précoce pour la réussite + [01:34:19][^7^][7] Formation et encadrement pédagogique des enseignants * Nécessité de former les enseignants à la pédagogie * Coordination entre l'enseignement secondaire et supérieur * Inclusion et co-enseignement dans les classes précaires + [01:37:04][^8^][8] Vision des étudiants et utilisation des nouvelles technologies * Importance de la réflexivité et du regard critique des enseignants * Utilisation des outils contemporains comme chat GPT dans l'enseignement * Adaptation de l'enseignement aux enjeux actuels et aux besoins des étudiants

Résumé de la vidéo [01:44:39][^1^][1] - [02:04:25][^2^][2] : La vidéo aborde la recherche-action "Lutter contre l'échec, repenser la relation pédagogique" et discute des défis de l'enseignement supérieur, notamment la nécessité d'adapter les méthodes pédagogiques à l'hétérogénéité des classes et l'importance de la formation des enseignants. Elle souligne également le rôle de l'enseignement supérieur dans la promotion de la justice sociale et l'égalité des chances, ainsi que les implications des examens d'entrée et du financement sur la réussite des étudiants.

Points saillants: + [01:44:39][^3^][3] Défis de l'enseignement supérieur * Adaptation aux grandes classes hétérogènes * Formation continue des enseignants * Impact des examens d'entrée sur la diversité étudiante + [01:47:29][^4^][4] Justice sociale et égalité des chances * Rôle de l'enseignement supérieur dans la réduction des inégalités * Importance du soutien aux étudiants sans antécédents universitaires * Nécessité d'un financement adéquat pour l'enseignement + [01:49:00][^5^][5] Financement et réforme de l'enseignement * Débat sur le refinancement de l'enseignement supérieur * Réforme des rythmes académiques et de la pédagogie * Propositions pour améliorer l'orientation et l'aide à la réussite + [01:59:02][^6^][6] Innovations pédagogiques et accueil des étudiants * Nouvelles approches pour l'accueil et l'intégration des étudiants en première année * Importance de l'auto-réflexion et de la compréhension du sens de l'éducation universitaire * Initiatives pour renforcer l'aide à la réussite et l'engagement étudiant

Résumé de la vidéo [02:04:27][^1^][1] - [02:06:15][^2^][2] :

La vidéo présente une discussion sur la fondation d'une université, soulignant l'importance de la diversité dans son conseil et la nécessité de financements privés pour compléter les fonds publics. Elle met en avant la collaboration avec des entreprises privées pour renforcer la position de l'université dans la région bruxelloise.

Points forts : + [02:04:27][^3^][3] Diversité du conseil * Représentation de la diversité étudiante * Membres variés comme Joseph Chovanek et Akima d'Armouche + [02:04:50][^4^][4] Financements privés et regard extérieur * Apport de fonds complémentaires * Challenge des méthodes et positionnement régional + [02:05:28][^5^][5] Fondation de l'université * Projet aligné sur les objectifs inclusifs * Booster complémentaire au soutien public + [02:05:44][^6^][6] Convergence politique * Accord sur les besoins et envies malgré les différences * Motivation collective pour relever les défis

Résumé de la vidéo [00:00:04][^1^][1] - [00:24:22][^2^][2] : La vidéo présente une journée d'étude sur l'expérience des jeunes aidants et des jeunes endeuillés. Elle aborde le cadre du dispositif "La vie, la mort, on en parle", initié au printemps 2021, qui est un portail de ressources sur la mort et le deuil pour les enfants et adolescents. La vidéo met en lumière l'importance de la recherche et de l'éducation sur ces sujets délicats.

Points forts : + [00:00:04][^3^][3] Introduction de la journée d'étude * Remerciements et contexte du dispositif "La vie, la mort, on en parle" * Présentation des objectifs et du programme de la journée + [00:02:01][^4^][4] Développement du dispositif de recherche * Lancement d'un dispositif de recherche sur la confrontation des jeunes à la finitude * Études sur la scolarisation des jeunes en situation palliative et des jeunes orphelins + [00:06:07][^5^][5] Présentation des recherches en cours * Focus sur les jeunes aidants endeuillés et l'impact du deuil sur la scolarité * Projets de recherche futurs et collaborations + [00:14:44][^6^][6] Intervention de partenaires et spécialistes * Contributions de la Fondation SIRP et de l'association Jeune Aident Ensemble * Importance du soutien et de la reconnaissance des jeunes orphelins

Résumé de la vidéo [00:24:24][^1^][1] - [00:47:01][^2^][2] : La vidéo présente une journée d'étude sur le vécu des jeunes aidants et des jeunes endeuillés. Elle explore les défis auxquels sont confrontés les enfants et les parents après la perte d'un proche, notamment en termes de scolarité et de soutien social.

Points forts : + [00:24:24][^3^][3] Impact sur les parents et la scolarité * Les parents partagent leurs stratégies pour aider leurs enfants à l'école * Certains informent l'école, d'autres changent d'école ou de maison * Les enfants peuvent être distraits ou avoir des comportements modifiés + [00:27:30][^4^][4] Perspective des enfants et changements observés * Les enfants expriment le manque, la tristesse et le vide ressenti * La perte affecte leur attention, concentration et comportement à l'école * Certains enfants se renferment ou ont des difficultés à suivre les cours + [00:30:45][^5^][5] Camarades comme ressources * Les amis peuvent devenir un soutien émotionnel important * Les enfants partagent des expériences similaires et renforcent les liens * L'identification et la proximité avec les pairs qui ont vécu des pertes similaires + [00:34:11][^6^][6] Conséquences à long terme et rôle des enseignants * La mort d'un parent a un impact sur la réussite scolaire et professionnelle * Les enseignants peuvent se sentir mal à l'aise et démunis face à la mort * Il est crucial de soutenir l'enfant et de créer une relation de confiance avec le parent

Résumé de la vidéo [00:47:03][^1^][1] - [01:07:56][^2^][2]:

La vidéo aborde les besoins des jeunes aidants et des jeunes endeuillés, en mettant l'accent sur la méconnaissance de ces besoins dans différents contextes sociaux et l'importance de la discussion et de la reconnaissance pour répondre à ces besoins.

Points forts: + [00:47:03][^3^][3] Méconnaissance des besoins * Manque de connaissance des besoins des enfants * Nécessité de discussions entre famille, école et loisirs * Importance de partager et d'exprimer les besoins + [00:52:01][^4^][4] Expérience des enseignants avec les orphelins * Les enseignants décrivent l'expérience comme déstabilisante * Ambivalence émotionnelle et besoin d'ajustement relationnel * Impact marquant sur les enseignants et les élèves + [00:57:01][^5^][5] Étude sur les représentations des enseignants * Exploration des attitudes des enseignants envers les jeunes orphelins * Manque de préparation et de formation pour gérer le deuil * Importance du soutien des collègues et des ressources disponibles

Résumé de la vidéo [01:07:57][^1^][1] - [01:31:51][^2^][2] : La vidéo présente une journée d'étude sur le vécu des jeunes aidants et des jeunes endeuillés. Elle aborde l'importance de travailler avec les familles, en particulier avec le parent restant, pour mieux comprendre et accompagner les enfants endeuillés. Les intervenants discutent de l'impact du deuil sur le développement et l'éducation des enfants, ainsi que de la nécessité d'une approche plus dynamique et intégrée pour soutenir les enfants et les familles touchées par le deuil.

Points saillants: + [01:08:00][^3^][3] Travailler avec les familles * Nécessité de collaboration avec le parent restant * Comprendre les comportements des enfants endeuillés * Prévenir les répercussions, notamment scolaires + [01:10:14][^4^][4] Anticipation de la prise en charge * Importance de l'anticipation dans les soins palliatifs * Impact de la préparation sur le deuil des enfants * Différences selon le type de décès et l'anticipation psychique + [01:20:00][^5^][5] Développement et éducation des enfants * Intégration de la dynamique de développement dans l'approche * Impact du deuil sur la trajectoire éducative des enfants * Nécessité d'opérationnaliser le modèle théorique pour la pratique + [01:26:13][^6^][6] Influence du milieu social sur le deuil * Conséquences socioéconomiques du deuil sur les familles * Variabilité de l'impact du deuil selon le milieu social * Relation entre le deuil et les facteurs sociaux et économiques

Résumé de la vidéo [01:31:53][^1^][1] - [01:55:59][^2^][2]:

Cette partie de la vidéo aborde la gestion du deuil chez les jeunes, en particulier dans le contexte scolaire. Les intervenants discutent des défis rencontrés par les enseignants et les professionnels de la santé pour soutenir les élèves endeuillés, ainsi que de l'importance de la communication et du soutien familial.

Points forts: + [01:32:00][^3^][3] La communication familiale * L'importance de rassembler la famille pour discuter * Les mécanismes de protection peuvent entraver la communication * Les entretiens familiaux permettent d'aborder les non-dits + [01:34:07][^4^][4] L'utilisation de la littérature jeunesse * Peut servir de média pour initier la conversation sur le deuil * Utile pour les échanges entre élèves et enseignants * Ne suffit pas seul, mais aide à structurer la parole + [01:34:39][^5^][5] Le rôle des enseignants * Souvent les premiers à prendre en charge l'orphelinage * La bienveillance des enseignants est cruciale * La temporalité du deuil peut affecter les apprentissages + [01:37:26][^6^][6] Le deuil à l'école * Le deuil fait partie de la vie de l'école * Nécessité de sensibiliser et former le personnel * L'impact du deuil sur les apprentissages est significatif + [01:45:01][^7^][7] La professionnalisation du personnel * Formation sur l'accompagnement du deuil en milieu scolaire * Intégration de la thématique dans l'adaptation à l'emploi * Élargissement de la formation à d'autres membres de la communauté scolaire + [01:50:07][^8^][8] Évaluation des pratiques professionnelles * Évaluation exploratoire sur l'accompagnement des jeunes en deuil * Importance de la formation pour réduire l'appréhension * Proposition de groupes de parole pour les élèves endeuillés

Résumé de la vidéo [01:56:01][^1^][1] - [02:01:05][^2^][2]:

Cette partie de la vidéo aborde l'expérience des jeunes aidants et endeuillés, mettant en lumière l'importance de reconnaître et d'intégrer la mort et le deuil dans le contexte scolaire. L'intervenant souligne la nécessité de former et de sensibiliser les éducateurs pour mieux accompagner les élèves dans leur parcours scolaire et de vie.

Points forts: + [01:56:01][^3^][3] L'importance de la formation * Nécessité d'augmenter les compétences * Importance de la persévérance malgré les défis financiers * Réinscription du programme dans le plan académique + [01:57:02][^4^][4] La mort et le deuil à l'école * Reconnaissance de la mort comme sujet scolaire * Importance de la sensibilisation et de la formation des éducateurs * Intégration de la mort dans le parcours de vie des élèves + [01:58:02][^5^][5] L'égalité des chances et l'inclusion * Inscription du programme dans les orientations ministérielles * Promotion de l'école inclusive pour tous les enfants * Accès à une scolarité diversifiée pour tous + [01:59:01][^6^][6] Sensibilisation des chefs d'établissement * Difficulté à engager les chefs d'établissement * Importance de la politique d'établissement pour la santé * Déploiement potentiel dans d'autres académies + [02:00:04][^7^][7] Accompagnement des élèves endeuillés * Mise en place de moyens supplémentaires pour l'accompagnement * Liaison avec les équipes des lycées pour un suivi continu * Objectif d'un accompagnement pérenne pour les élèves

Résumé de la vidéo [00:00:04][^1^][1] - [00:23:49][^2^][2]:

Cette vidéo présente la deuxième partie d'une journée d'étude sur l'expérience des jeunes aidants et des jeunes endeuillés. Elle se concentre sur les défis et les impacts de ces rôles sur le bien-être et la scolarité des jeunes.

Points forts: + [00:00:04][^3^][3] Définition et rôle des jeunes aidants * Un jeune aidant est un enfant ou adolescent qui aide régulièrement un membre de sa famille souffrant d'une maladie ou d'un handicap. * Ils peuvent effectuer des tâches ménagères, administratives, ou fournir un soutien moral et des soins personnels. * L'aide apportée est évaluée sur un continuum de faible à très importante. + [00:04:37][^4^][4] Évolution de la reconnaissance des jeunes aidants en France * La prise de conscience en France a commencé en 2014, avec des initiatives clés et la création de l'association nationale Jeunes Aidants Ensemble. * En 2019, le gouvernement a identifié les jeunes aidants dans la stratégie "Agir pour les aidants". + [00:07:05][^5^][5] Recherche sur les jeunes aidants * Un programme de recherche vise à identifier les jeunes aidants en France, étudier les facteurs de protection et de vulnérabilité, et développer des interventions pour les aider. * Des études ont été menées pour comprendre les caractéristiques, les besoins et les difficultés des jeunes aidants. + [00:20:26][^6^][6] Conséquences sur la santé et la scolarité * Les jeunes aidants rapportent une moins bonne qualité de vie et ont souvent eux-mêmes des problèmes de santé. * Ils ont tendance à redoubler plus souvent et à choisir des formations à distance pour rester proches de leurs proches aidés.

Résumé de la vidéo [00:21:00][^1^][1] - [01:40:17][^2^][2]:

Cette partie de la vidéo se concentre sur les jeunes aidants en France, leur identification, les défis auxquels ils sont confrontés et les efforts pour développer des interventions de soutien. La présentation souligne l'importance de reconnaître et d'accompagner ces jeunes, qui fournissent souvent des soins à un membre de la famille souffrant de maladie ou de handicap.

Points forts: + [00:21:00][^3^][3] Recherche sur les jeunes aidants * Identification des jeunes aidants en France * Étude des facteurs de protection et de vulnérabilité * Développement d'interventions pour les soutenir + [00:41:00][^4^][4] Limites de la recherche actuelle * Échantillon limité ne permettant pas de généraliser * Manque d'informations sur le contexte scolaire des jeunes + [00:57:00][^5^][5] Expérience dans une école d'ingénieurs * Mise en place d'entretiens pour comprendre les besoins des étudiants endeuillés * Adaptation des aménagements scolaires en fonction des situations individuelles + [01:18:00][^6^][6] Difficultés systémiques dans l'éducation * L'épuisement des jeunes aidants affecte leur capacité à s'engager dans les démarches administratives * Besoin d'un soutien pour naviguer dans les processus institutionnels

Résumé de la vidéo [01:21:00][^1^][1] - [01:23:09][^2^][2]:

Cette partie de la vidéo se concentre sur les résultats d'une étude qualitative exploratoire en psychologie, nommée "jadisp", qui examine la perception des interférences entre le processus de deuil et la scolarité chez les jeunes aidants. L'étude a été soutenue financièrement par la Fondation Osirp et vise à comprendre les conséquences de l'apport des jeunes aidants, en particulier sur leur scolarité.

Points forts: + [01:21:00][^3^][3] Présentation de l'étude jadisp * Recherche qualitative exploratoire en psychologie * Soutien financier de la Fondation Osirp * Focus sur les jeunes aidants et le deuil + [01:22:18][^4^][4] Définition des jeunes aidants * Clarification sur l'apport des jeunes aidants * Conséquences multiples, y compris sur la scolarité * Importance de la prise en compte de ces incidences + [01:22:55][^5^][5] Résultats de l'étude * Interférences entre deuil et scolarité * Perception des jeunes aidants sur ces interférences * Objectif de mieux comprendre et accompagner les jeunes aidants

1 стадия изучения 2 влюбленность подкреплять стабильность. и классное в 1 момент нужно сделать легкую яму 3.начало любви

The lopsided asymmetry here is charring. Imagine putting in time like that, where responses are instantly generated. You'll feel a) that this is worth something because you spent time on this, and we equate such investment in others with depth, but here there's no other b) there will always be a response by generated personas, and you will feel a likely 'social' pressure to respond in kind.

And of course there's lots of money in this sociopathic scheme. likely because the funders come from the same strain.

wholesome? 'could help people connect with others such as AIs' An AI is not an other and there is no connection as it has no concept nor memory of you.

It might well be a narcissist honey pot.

Where on the spectrum is this guy? No they couldn't have been AIs, or you wouldn't have built meaningful connections. Relations aren't just something you experience for you to feel good over, but a thing in itself playing out between two or more people. This sounds sociopathic.

Will humans and AI be distinguishable? _butterfly who's a woodworker' no it isn't. It is generating stuff statistically matching what a woodworker might post, and all it generates is fantasy. There is no woodworker, there is no 'latest creation' of actual wood, just a generated image. Can we please stop this utter crap?

whose creativity? what actual relationships? More seductive to project you mean.

Yet this too is just a generative AI chatbot, with nice pictures.

What? a Butterfly is an interactive persona, and supposedly people want to interact with it? Each one has "backstories, opinions and emotions" no they don't. Each one generates text that people then project upon to perceive a past, opinions and emotions. It even doesn't need much for it, see Eliza.

These descriptions are very uncanny valley. Imagine a community where each AI friend has its own unique digital life, ready to share moments, create memories, post images just like real friends Butterflies is more than just a social network; it’s a fresh approach to connection Imagine a place where every friend understands you perfectly,

eLife assessment

This important study examines the relationship between expiratory airflow and vocal pitch in adult mice during the production of ultrasonic vocalizations and also identifies a molecularly defined population of brainstem neurons that regulates mouse vocal production across development. The evidence supporting the study's conclusions that expiratory airflow shapes vocal pitch and that these brainstem neurons preferentially regulate expiratory airflow is novel and compelling. This work will be of interest to neuroscientists working on mechanisms and brainstem circuits that regulate vocal production and vocal-respiratory coordination.

Reviewer #1 (Public Review):

Summary:

In this important work, the authors propose and test a model for the control of murine ultrasonic vocalizations (USV) in which two independent mechanisms involving changes in laryngeal opening or airflow control vocal tone. They present compelling experimental evidence for this dual control model by demonstrating the ability of freely behaving adult mice to generate vocalizations with various intonations by modulating both the breathing pattern and the laryngeal muscles. They also present novel evidence that these mechanisms are encoded in the brainstem vocalization central neural pattern generator, particularly in the component in the medulla called the intermediate reticular oscillator (iRO). The results presented clearly advance understanding of the developmental nature of the iRO, its ability to intrinsically generate and control many of the dynamic features of USV, including those related to intonation, and its coordination with/control of expiratory airflow patterns. This work will interest neuroscientists investigating the neural generation and control of vocalization, breathing, and more generally, neuromotor control mechanisms.

Strengths:

Important features and novelty of this work include:

(1) The study employs an effective combination of anatomical, molecular, and functional/ behavioral approaches to examine the hypothesis and provide novel data indicating that expiratory airflow variations can change adult murine USV's pitch patterns.

(2) The results significantly extend the authors' previous work that identified the iRO in neonatal mice by now presenting data that functionally demonstrates the existence of the critical Penk+Vglut2+ iRO neurons in adult mice, indicating that the iRO neurons maintain their function in generating vocalization throughout development.

(3) The results convincingly demonstrate that the iRO neurons encode and can generate vocalizations by modulating both breathing and the laryngeal muscles.

(4) The anatomical mapping and tracing results establish an important set of input and output circuit connections to the iRO, including input from the vocalization-promoting subregions of the midbrain periaqueductal gray (PAG), as well as output axonal projections to laryngeal motoneurons, and to the respiratory rhythm generator in the preBötzinger complex.

(5) These studies advance the important concept that the brainstem vocalization pattern generator integrates with the medullary respiratory pattern generator to control expiratory airflow, a key mechanism for producing various USV types characterized by different pitch patterns.

Weaknesses:

A limitation is that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow has not been fully worked out, requiring future studies.

Reviewer #2 (Public Review):

Summary:

Both human and non-human animals modulate the frequency of their vocalizations to communicate important information about context and internal state. While regulation of the size of the laryngeal opening is a well-established mechanism to regulate vocal pitch, the contribution of expiratory airflow to vocal pitch is less clear. To consider this question, this study first characterizes the relationship between the dominant frequency contours of adult mouse ultrasonic vocalizations (USVs) and expiratory airflow using whole-body plethysmography. The authors also include data from a single mouse that combines EMG recordings from the diaphragm and larynx with plethysmography to provide evidence that the respiratory central pattern generator can be re-engaged to drive "mini-breaths" that occur during the expiratory phase of a vocal breath. Next, the authors build off of their previous work characterizing intermediate reticular oscillator (iRO) neurons in mouse pups to establish the existence of a genetically similar population of neurons in adults and show that artificial activation of iRO neurons elicits USV production in adults. Third, the authors examine the acoustic features of USV elicited by optogenetic activation of iRO and find that a majority of natural USV types (as defined by pitch contour) are elicited by iRO activation and that these artificially elicited USVs are more likely than natural USVs to be marked by positive intonation (positive relationship between USV dominant frequency and expiratory airflow).

Strengths:

Strengths of the study include the novel consideration of expiratory airflow as a mechanism to regulate vocal pitch and the use of intersectional methods to identify and activate the iRO in adult mice. The establishment of iRO neurons as a brainstem population that regulates vocal production across development is an important finding.

Weaknesses:

The conclusion that the respiratory CPG is re-engaged during "mini-breaths" throughout a given vocal breath would be strengthened by including analyses from more than one mouse.

Author response:

The following is the authors’ response to the original reviews.

In the revised manuscript we have included an additional study that significantly contributes to the conclusions and models of the original version. Briefly, Figure 3 now describes our characterization of the diaphragm and laryngeal muscle activities (electromyography, EMG) during endogenous vocalizations. These EMGs also serve as representations of the brainstem breathing central pattern generator (CPG) inspiratory and post-inspiratory generating neurons, respectively. In our original submission, we found that many of the vocalizations had changes in pitch that mirrored the change in expiratory airflow (we termed positive intonation), and we proposed that the coordination of breathing muscles (like the inspiratory muscles) and larynx patterned this. This mechanism is akin to our findings for how neonatal cries are rhythmically timed and produced (Wei et al. 2022). The newly presented EMG data re-inforces this idea. We found that for vocalizations with positive intonation, the inspiratory diaphragm muscle has an ectopic burst(s) of activity during the expiration phase which corresponds to a decrease in airflow and pitch, and this is followed by laryngeal muscle activity and increased pitch. This can be cycled throughout the expiration to produce complex vocalizations with oscillations in pitch. A basal breath is hardwired for the laryngeal muscle activity to follow the diaphragm, so the re-cycling of this pattern nested within an expiration (a ‘mini-breath’ in a ‘breath’) demonstrates that the vocalization patterning system engages the entire breathing CPG. This contrasts with the canonical model that activity of the laryngeal premotor neurons control all aspects of producing / patterning vocalizations. Furthermore, this mechanism is exactly how the iRO produces and patterns neonatal vocalizations (Wei et al. 2022) and motivates the likely use of the iRO in adult vocalizations.

Response to recommendations for the authors:

Reviewer #1:

(1) The authors should note in the Discussion that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow have not been fully worked out, requiring future studies.

This was noted in the discussion section “The iRO likely patterns intonation for endogenous phonation”.

(2) Please change the labeling of the last supplemental figure to Figure Supplemental 5.

Thank you for identifying this.

Reviewer #2:

Major concerns

(1) While it is true that modulation of activity in RAm modulates the laryngeal opening, this statement is an incomplete summary of prior work. Previous studies (Hartmann et al., 2020; Zhang et al., 1992, 1995) found that activation of RAm elicits not just laryngeal adduction but also the production of vocal sounds, albeit vocal sounds that were spectrally dissimilar from speciestypical vocalizations. Moreover, a recent study/preprint that used an activity-dependent labeling approach in mice to optogenetically activate RAm neurons that were active during USV production found that re-activation of these neurons elicits USVs that are acoustically similar to natural USVs (Park et al., 2023). While the authors might not be required to cite that recent preprint (as it is not yet peer-reviewed), the fact that activation of RAm elicits vocal sounds is clear evidence that its effects go beyond modulating the size of the laryngeal opening, as this alone would not result in sound production (i.e., RAm activation must also recruit expiratory airflow). The authors should include these relevant studies in their Introduction. Moreover, the rationale for the model proposed by the authors (that RAm controls laryngeal opening whereas iRO controls expiratory airflow) is unclear with regard to these prior studies. The authors should include a discussion of how these prior findings are consistent with their model (as presented in the Introduction, as well as in Figure 4 and relevant Discussion) that RAm modulates the size of laryngeal opening but not expiratory airflow.

An introduction and discussion of the Veerakumar et. al. 2023 and Park et. al. 2024 manuscripts describing RAm in mice has now been included.

The iRO serves to coordinate the breath airflow and laryngeal adduction to produce sound and the intonation within it that mirrors the breath airflow. This occurs because the iRO can control the breathing CPG (synaptic input to the preBötC inspiratory pacemaker) and is premotor to multiple laryngeal muscles (Wei et. al. 2022). The modulation of the expiratory airflow is by inducing momentary contraction of the diaphragm (via excitation of the preBötC) which opposes (a.k.a. slows) expiration. This change in flow results in a decrease in pitch (Fig. 3 in the revised manuscript, Wei et. al. 2022).

It is our understanding that the basic model for RAm evoked USVs is that RAm evokes laryngeal adduction (and presumed abdominal expiratory muscle activation) and this activity is momentarily stopped during the breath inspiration by inhibition from the preBötC (Park et. al. 2024). So, in this basic model, any change in pitch and expiratory airflow would be controlled by tuning RAm activity (i.e., extent of laryngeal adduction). In this case, the iRO induced inspiratory muscle activity should not occur during expiration, which is not so (Fig. 3). Note, the activity of abdominal expiratory muscles during endogenous and RAm evoked USVs has not been characterized, so the contribution of active expiration remains uncertain. This is an important next step.

We have now included a discussion of this topic which emphasizes that iRO and RAm likely have reciprocal interactions (supported by the evidence of this anatomical structure). These interactions would explain why excitation of either group can evoke USVs and, perhaps, the extent that either group contributes to a USV explains how the pitch / airflow changes. An important future experiment will be to determine the sufficiency of each site in the absence of the other.

(2) The authors provide evidence that the relationship between expiratory airflow and USV pitch is variable (sometimes positive, sometimes negative, and sometimes not related). While the representative spectrograms clearly show examples of all three relationship types, no statistical analyses are included to evaluate whether the relationship between expiratory airflow and USV pitch is different than what one would expect by chance. For example, if USV pitch were actually unrelated to expiratory airflow, one might nonetheless expect spurious periods of positive and negative relationships. The lack of statistical analyses to explicitly compare the observed data to a null model makes it difficult to fully evaluate to what extent the evidence provided by the authors supports their claims.

We have now included two null distributions and compared our observed correlation values to these. The two distributions were created by taking each USV / airflow pair and randomly shuffling either the normalized USV pitch values (pitch shuffled) or the normalized airflow values (airflow shuffled) to simulate the distribution of data should no relationship exist between the USV pitch and airflow.

(3) The relationship between expiratory airflow and USV pitch comes with two important caveats that should be described in the manuscript. First, even in USV types with an overall positive relationship between expiratory airflow and pitch contour, the relationship appears to be relative rather than absolute. For example, in Fig. 2E, both the second and third portions of the illustrated two-step USV have a positive relationship (pitch goes down as expiratory airflow goes down). Nonetheless, the absolute pitch of the third portion of that USV is higher than the second portion, and yet the absolute expiratory airflow is lower. The authors should include an analysis or description of whether the relationship between expiratory airflow and USV pitch is relative vs.

absolute during periods of 'positive intonation'.

The relationship between pitch and airflow is relative and this in now clarified in the text. To determine this, we visualized the relationship between the two variables by scatterplot for each of the USVs syllables and, as the reviewer notes, a given airflow cannot predict the resulting frequency and vice versa.

(4) A second important caveat of the relationship between expiratory airflow and USV pitch is  that changes in expiratory airflow do not appear to account for the pitch jumps that characterize mouse USVs (this lack of relationship also seems clear from the example shown in Fig. 2E). This caveat should also be stated explicitly.

The pitch jumps do not have a corresponding fluctuation in airflow, and this is now stated in the results and discussion.

(5) The authors report that the mode of relationship between expiratory airflow and USV pitch (positive intonation, negative intonation, or no relationship) can change within a single USV. Have the authors considered/analyzed whether the timing of such changes in the mode of relationship coincides with pitch jumps? Perhaps this isn’t the case, but consideration of the question would be a valuable addition to the manuscript.

We analyzed a subset of USVs with pitch jumps that were defined by a change >10 kHz, at least 5ms long, and had one or two jumps. The intonation relationships between the sub-syllables within a USV type were not stereotyped as evidenced by the same syllable being composed of combinations of both modes.

(6) The authors incorrectly state that PAG neurons important for USV production have been localized to the ventrolateral PAG. Tschida et al., 2019 report that PAG-USV neurons are located predominantly in the lateral PAG and to a lesser extent in the ventrolateral PAG (see Fig. 5A from that paper). The finding that iRO neurons receive input from VGlut2+ ventrolateral PAG neurons represents somewhat weak evidence that these neurons reside downstream of PAG-USV neurons. This claim would be strengthened by the inclusion of FOS staining (following USV production), to assess whether the Vglut+ ventrolateral PAG neurons that provide input to iRO are active in association with USV production.

This comment correctly critiques that our PAG à iRO tracing does not demonstrate that the labeled PAG neurons are sufficient nor necessary for vocalization. Directly demonstrating that activation and inhibition the PAG-iRO labeled neurons ectopically drives or prevents endogenous USVs is an important next step. While FOS implies this connectivity, it does not definitely establish it and so this experiment is impacted by some of the caveats of our tracing (e.g. PAG neurons that drive sniffing might be erroneously attributed to vocalization).

Our reading of the literature could not identify an exact anatomical location within the mouse PAG and this site appears to vary within a study and between independent studies (like within and between Tschida et. al. 2019 and Chen et. al. 2021). The labeling we observed aligns with some examples provided in these manuscripts and with the data reported for the retrograde tracing from RAm (Tschida et al 2019).

(7) In Figure S5A, the authors show that USVs are elicited by optogenetic activation of iRO neurons during periods of expiration. In that spectrogram, it also appears that vocalizations were elicited during inspiration. Are these the broadband vocalizations that the authors refer to in the Results? Regardless, if optogenetic activation of iRO neurons in some cases elicits vocalization both during inspiration and during expiration, this should be described and analyzed in the manuscript.

The sound observed on the spectrogram during inspiration is an artefact of laser evoked head movements that resulted in the fiber cable colliding with the plethysmography chamber. In fact, tapping an empty chamber yields the same broad band spectrogram signal. The evoked USV or harmonic band vocalization is distinct from this artefact and highlighted in pink.

(8) Related to the comment above, the authors mention briefly that iRO activation can elicit broadband vocalizations, but no details are provided. The authors should provide a more detailed account of this finding.

The broadband harmonic vocalizations we sometimes observe upon optogenetic stimulation of AAV-ChR2 expressing iRO neurons are akin to those previously described within the mouse vocal repertoire (see Grimsley et. al .2011). We have added this citation and mentioned this within the text. 

(9) The effects of iRO stimulation differ in a couple of interesting ways from the effects of PAGUSV activation. Optogenetic activation of PAG-USV neurons was not found to entrain respiration or to alter the ongoing respiratory rate and instead resulted in the elicitation of USVs at times when laser stimulation overlapped with expiration. In contrast, iRO stimulation increases and entrains respiratory rate, increases expiratory and inspiratory airflow, and elicits USV production (and also potentially vocalization during inspiration, as queried in the comment above). It would be informative for the authors to add some discussion/interpretation of these differences.

We have added a section of discussion to describe the how these different results may be explained by the iRO being a vocal pattern generator versus the PAG as a ‘gating’ signal to turn on the medullary vocalization patterning system (iRO and RAm). See discussion section ‘The iRO likely patterns intonation for endogenous phonation’.

(10) The analysis shown in Fig. 4D is not sufficient to support the author’s conclusion that all USV types elicited by iRO activation are biased to have more positive relationships between pitch and expiratory airflow. The increase in the relative abundance of down fm USVs in the opto condition could account for the average increase in positive relationship when this relationship is considered across all USV types in a pooled fashion. The authors should consider whether each USV type exhibits a positive bias. Although such a comparison is shown visually in Fig. 4G, no statistics are provided. All 7 USV types elicited by optogenetic activation of iRO should be considered collectively in this analysis (rather than only the 5 types currently plotted in Fig. 4G).

In the original submission the statistical analysis of r values between opto and endogenous conditions was included in the figure legend (‘panels E-G, two-way ANOVA with Sidak’s post-hoc test for two-way comparisons was used; all p-values > 0.05), and this has not changed in the revised manuscript. We have now provided the suggested comparison of opto vs endogenous USVs without down fm (Fig. 5D). This positive shift in r is statistically significant (…).

(11) The evidence that supports the author’s model that iRO preferentially regulates airflow and that RAm preferentially regulates laryngeal adduction is unclear. The current study finds that activation of iRO increases expiratory (and inspiratory) airflow and also elicits USVs, which means that iRO activation must also recruit laryngeal adduction to some extent. As the authors hypothesize, this could be achieved by recruitment of RAm through iRO’s axonal projections to that region.

Note, it is more likely that iRO is directly recruiting laryngeal adduction as they are premotor to multiple laryngeal muscles like the thyroarytenoid and cricothyroid (Wei et. al. 2022). The ‘Discussion’ now includes our ideas for how the iRO and RAm likely interact to produce vocalizations.

In the recent preprint from Fan Wang’s group (Park et al., 2023), those authors report that RAm is required for USV production in adults, and that activation of RAm elicits USVs that appear species-typical in their acoustic features and elicits laryngeal adduction (assessed directly via camera). Because RAm activation elicits USVs, though, it must by definition also recruits expiratory airflow. Can the authors add additional clarification of how the evidence at hand supports this distinction in function for iRO vs RAm?

See response to ‘Major Concern #1”.

Minor concerns 

(1) The authors might consider modifying the manuscript title. At present, it primarily reflects the experiments in Figure 2.

We have provided a title that we feel best reflects the major point of the manuscript. We hope that this simplicity enables it to be recognized by a broad audience of neuroscientists as well as specialists in vocalization and language.

(2) The statement in the abstract that "patterns of pitch are used to create distinct 'words' is somewhat unclear. Distinct words are by and large defined by combinations of distinct phonemes. Are the authors referring to the use of "tonemes" in tonal languages? If so, a bit more explanation could be added to clarify this idea. This minor concern includes both the Abstract, as well as the first paragraph of the Introduction.

We have clarified this line in the abstract to avoid the confusing comparison between mouse vocalizations and human speech. In the introduction we have expanded our explanation to clarify that variations in pitch are a component of spoken language that add additional meaning and depth to the underlying, phonemic structure. 

(3) Multiple terms are used throughout the manuscript to refer to expiratory airflow: breath shape (in the title), breath pattern, deviations in exhalation, power of exhalation, exhalation strength, etc. Some of these terms are vague in meaning, and a consolidation of the language would improve the readability of the abstract and introduction.

We have chosen a smaller selection of descriptive words to use when describing these breath features.

(4) Similarly, "exhalation" and "expiration" are both used, and a consistent use of one term would help readability.

See point 3.

(5) In a couple of places in the manuscript, the authors seem to state that RAm contains both laryngeal premotor neurons as well as laryngeal motor neurons. This is not correct to our knowledge., but if we are mistaken, we would ask that the authors add the relevant references that report this finding.

It is our understanding that the RAm is defined as the anatomical region consistent with the murine rostral and caudal ventral respiratory groups composed of multiple premotor neuron pools to inspiratory, expiratory, laryngeal, and other orofacial muscles. This is supported by neurons within RAm that reflect multiple phases of the inspiratory and expiratory cycle (Subramanian et. al. 2018) and excitation of sub-regions within RAm modulating multiple parts of the breathing control system (Subramanian et. al. 2018 and Subramanian 2009). Rabies tracing of the various premotor neurons which define the anatomical region of RAm in the mouse shows that they surround the motor neurons in the loose region of the nucleus ambiguus (the anatomical location of RAm) for multiple muscles of the upper airway system, such as the thyroarytenoid (Wu et. al. 2017, Dempsey et. al. 2021 and Wei et. al. 2022). Given that the name RAm reflects a broad anatomical location, we have used it to describe both the premotor and motor neurons embedded within it. We have now clarified this in the text.

(6) The statistical analysis applied in Figure 1C is somewhat confusing. The authors show two distributions that appear different but report a p-value of 0.98. Was the analysis performed on the mean value of the distributions for each animal, the median, etc.? If each animal has two values (one for USV+ breaths and one for USV- breaths), why not instead compare those with a paired t-test (or Wilcoxon rank sign)? Additional information is needed to understand how this analysis was performed.

The original manuscript version used a two-way anova to compare the normalized histogram of instantaneous frequency for breaths with (USV+) or without (USV-) for each animal (first factor: USV+/-, second factor: Frequency). The p-value for the first factor (USV) was 0.98 showing no statistically significant effect of USV on the distribution of the histogram.

For simplicity, we have instead performed the analysis as suggested and include a bar graph. This analysis shows that the instantaneous frequency of USV breaths is, in fact, statistically significantly lower than those without USVs. We have updated the figure legend and text to reflect this.

(7) The use of the word "syllable" to describe parts of a USV that are produced on a single breath may be confusing to some scientists working on rodent USVs. The term 'syllable' is typically used to describe the entirety of a USV, and the authors appear to use the term to describe parts of a USV that are separated by pitch jumps. The authors might consider calling these parts of USVs "sub-syllables".

We have clarified these descriptions throughout the text. We now refer to the categories as ‘syllable types’, define ‘syllables’ as ‘a continuous USV event’ with no more than 20ms of silence within and finally ‘sub-syllables’ to refer to components of the syllable separated by jumps in frequency (but not gaps in time).

(8) In Figure S3, final row, the authors show a USV produced on a single breath that contains two components separated by a silent period. This type of bi-syllabic USV may be rare in adults and is similar to what the authors showed in their previous work in pups (multiple USVs produced on a single expiration, separated by mini-inspirations). One might assume that the appearance of such USVs in pups and their later reduction in frequency represents a maturation of vocalrespiratory coordination. Nonetheless, the appearance of bi-syllabic USVs has not been reported in adult mice to our knowledge, and the authors might consider further highlighting this finding.

We were also struck by the similarity of these USVs to our study in neonates and such types of similarities sparked an interest in the role of the iRO in patterning adult USVs. We now include a description of the presence and abundance of bi- and tri-syllablic calls observed in our recordings to highlight this finding.

(9) Figure 4 is referenced at the end of the second Results section, but it would seem that the authors intended to reference Figure 2. 

For simplicity we included some of the referenced data within Fig. S5. We appreciate the recommendation.

(10) In the optogenetic stimulation experiments, the authors should clarify why bilateral stimulation was applied. Was unilateral stimulation ineffective or less effective? The rationale provided for the use of bilateral stimulation (to further localize neural activation) is unclear.

The iRO is bilateral and, we presume, functions similarly. So, we attempted to maximally stimulate the system. We have clarified this in the methods.

(11) Figure Supplemental '6' should be '5'.

Thanks!

(12) Last sentence of the Introduction: "Lasty" should be "lastly".

Thanks!

(13) There are two references for Hage et al., 2009. These should be distinguished as 2009a and 2009b for clarity.

Thanks!

please bold prompt

Author response:

The following is the authors’ response to the current reviews.

We thank the reviewers and editor for their careful review of our work. We believe the resulting manuscript is much stronger. We agree with the comments made by Reviewer #2 regarding additional histology and neuronal data analysis, which will be presented in subsequent work.

The following is the authors’ response to the original reviews.

Reviewer 1 (Public Weaknesses):

It was not always clear what the lesion size was. This information is important for future applica- tions, for example, in the visual cortex, where neurons are organized in retinotopy patterns.

We thank the reviewer for this feedback. While there is some variation in lesion volume for a given parameter set, we have added more details of the volumes of lesions created in our testing (Fig. 4 and Fig. 5).

It would be helpful if the author could add some discussion about whether and how this method could be used in other types of array/multi-contact electrodes, such as passive neuropixels, S- probes, and so on. In addition, though an op-amp was used in the design, it would still be helpful if the author could provide a recommended range for the impedance of the electrodes.

We thank the reviewer for this suggestion. We have both added a demonstration of use in a differ- ent multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

“We demonstrated that our electrolytic lesioning technique works with a linear multicontact probe by testing with a U-Probe in ex vivo rabbit cortex. There are no particular limitations that would prevent our specific electrolytic lesioning technique and device from working with any passive multielectrode probe. The main requirements for use are that the probe has two electrodes that can directly (via whatever necessary adapters) connect to the lesioning device, such that arbitrary current can be passed into them as the anode and cathode. This would limit use of probes, like Neuropixels, where the on-chip acquisition and digitization circuitry generally precludes direct connection to electrodes [1], [2]. The impedance of the multielectrode probe should not be an issue, due to the use of an op amp. We showed use  with a Utah array (20-800 kΩ) and a U-Probe (1-1.5 MΩ). The specific op amp used here has a voltage range of ± 450 V, which assuming a desired output of 150 µA of current would limit electrode impedance to 6 MΩ. Though a different op amp could easily be used to accommodate a higher electrode impedance, it is unlikely that this would be necessary, since most electrodes have impedances between 100 kΩ to 1 MΩ [3].”

Reviewer 2 (Public Weaknesses):

In many of the figures, it is not clear what is shown and the analysis techniques are not well described.

We thank the reviewer for this feedback. We hope that our edits to both the figures and the text have improved clarity for readers.

The flexibility of lesioning/termination location is limited to the implantation site of the multielec- trode array, and thus less flexible compared to some of the other termination methods outlined in Appendix 2.

We thank the reviewer for this point. You are right that the lesioning location is limited to the multielectrode array’s implantation site, while other methods in Appendix 2 do not require prox- imity of the lesion location and the electrophysiology recording site. However, we believe that the closeness of the lesioning location to the microelectrode array is a strength - guaranteeing record- ings from the perilesional area - even with the small negative of reduced flexibility. Multielectrode arrays can be implanted in many areas of cortex. If one wanted to study distal effects of a lesion, additional electrophysiology probes could be implanted to record from those areas. We have noted this on Page 3, Line 117.

“While the link between the lesion location and the multielectrode location technically con- strains the lesion to an area of cortex in which a multielectrode array could be implanted, we see the connection as a positive, because it ensures recording some neuroelectrophysiology from the perilesional area in which recovery is hypothesized to occur (see Appendix 1Data Availabilityappendix.41).”

Although the extent of the damage created through the Utah array will vary based on anatomical structures, it is unclear what is the range of lesion volumes that can be created with this method, given a parameter set. It was also mentioned that they performed a non-exhaustive parameter search for the applied current amplitude and duration (Table S1/S2) to generate the most suitable lesion size but did not present the resulting lesion sizes from these parameter sets listed. Moreover, there’s a lack of histological data suggesting that the lesion size is precise and repeatable given the same current duration/amplitude, at the same location.

We thank the reviewer for this thoughtful feedback. We have added figures (Figs. 4 and 5), where we show the relationship between estimated lesion volume and the current amplitude and duration parameters. These figures include more data from the tests in Supplementary File 1 and Supplementary File 2. While there is some variation in lesion volume for a given current amplitude and duration, there is still a clear relationship between the parameters and lesion volume.

It is unclear what type of behavioral deficits can result from an electrolytic lesion this size and type (∼3 mm in diameter) in rhesus macaques, as the extent of the neuronal loss within the damaged parenchyma can be different from past lesioning studies.

While we appreciate the reviewer’s interest in the behavioral deficits associated with our lesions in rhesus macaques, reporting these falls beyond the scope of this manuscript. Future work will explore the behavioral deficits associated with these lesions

The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study.

We apologize for not being more explicit about the parameters used for the lesion in Monkey F. We have added this in Results on Page 5, Line 209 and in Methods on Page 19, Line 586.

“After this validation and refinement, one proof-of-concept lesion (150 µA direct current passed through adjacent electrodes for 45 seconds) was performed in an in vivo sedated rhe- sus macaque (Monkey F) in order to validate the safety of the procedure.”

“This lesion was created by applying 150 µA of direct current to two adjacent electrodes in the microelectrode array for 45 seconds.”

We also clarified the parameters used for the other lesions in Monkeys H and U in Results on Page 7, Line 233 and in Methods on Page 19, Line 586.

“In all of the fourteen lesions across two awake-behaving rhesus macaques (150 µA direct current passed through adjacent electrodes for 30 or 45 seconds (30s for Monkey U and 45s for Monkey H, except lesion H200120 which was for 50 seconds)), the current source worked as expected, providing a constant current throughout the duration of the procedure.”

“In these lesions, 150 µA of direct current was applied to two adjacent electrodes in the mi- croelectrode array for 30 or 45 seconds (30s for Monkey U, 45s for Monkey H), except in lesion H200120 where current was applied for 50 seconds.”

Monkey F was euthanized shortly after the lesion, so we now mention this on Page 19, Line 583.

“Based on this, and a lack of physiological signs of pain from the anaesthetized pig studies, a lesion was performed on a sedated rhesus macaque who was subsequently euthanized due to unrelated health complications (Monkey F; 16 year-old adult, male rhesus macaque) in order to further verify safety before use in awake-behaving rhesus.”

Because Monkey F was sedated and then euthanized shortly after, there is no behavioral data. As the lesion in sedated Monkey F was used to validate the safety of the procedure, any further data and analysis fall beyond the scope of this manuscript.

As an inactivation method, the electrophysiology recording in Figure 5 only showed a change in pairwise comparisons of clustered action potential waveforms at each electrode (%match) but not a direct measure of neuronal pre and post-lesioning. More evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning. Some exam- ples of this can be showing the number of spike clusters identified each day, as well as analyzing local field potential and multi-unit activity.

The reviewer has pointed out some short comings of the original analysis, which we believe have since been addressed with the revised analysis. LFP and spiking activity are functional measures that are more ambiguous in terms of loss and are also the subject of another manuscript currently under revision.

The advantages over recently developed lesioning techniques are not clear and are not discussed.

We thank the reviewer for noting this. We have added a section, also responding to their later request for us to compare our work to Khateeb et al. 2022, by adding a section to the Discussion on Page 16, Line 434.

“Perhaps the most unique advantage of our technique in comparison with other existing inactivation methods lies in Design Consideration #1: stable electrophysiology pre- and post-inactivation (Appendix 1Data Availabilityappendix.41). While several methods exist that allow for localization and size control of the inactivation (Design Consideration #2) and cross compatibility across regions and species (Design Consideration #3), few have achieved compatibility with stable electrophysiology. For example, some studies record electrophysiology only after the creation of the lesion, preventing comparison with baseline neuronal activity [4]. One recent study, Khateeb, et al., 2022, developed an inactivation method that is effectively combined with stable electrophysiology by creating photothrombotic lesions through a chronic cranial window integrated with an electrocorticography (ECoG) array [5], which may be appropriate for applications where local field potential (LFP) recording is sufficient. This approach has trade-offs with regards to the three design considerations presented in Appendix 1Data Availabilityappendix.41.

While Khateeb, et al., present a toolbox with integrated, stable electrophysiology from an ECoG array pre- and post- inactivation (Design Consideration #1), it demonstrated recordings from an ECoG array with limited spatial resolution. While a higher density ECoG array that would provide higher spatial resolution could be used, increasing the density of opaque electrodes might occlude optical penetration and constrain photothrombotic lesions. Further, ECoG arrays are limited to recording LFP, not electrophysiology at single neuron resolution, potentially missing meaningful changes in the neuronal population activity after lesioning. Khateeb, et al., demonstrated localization and control the size of inactivation (Design Consideration #2). In this manuscript, we have shown that the amount and duration of direct current are significant determinants of lesion size and shape, while with photothrombotic lesions, light intensity and aperture diameter are the significantly relevant parameters. One potential advantage of photothrombotic approaches is the use of optical tools to monitor anatomical and physiological changes after lesioning through the cranial window, though the research utility of this monitoring remains to be demonstrated.

Although the method presented by Khateeb, et al., shows some cross-compatibility (Design Consideration #3), it has greater limitations in comparison with the method presented here. For example, while Khateeb, et al., notes that the approach could be adapted for use in smaller organisms, no modification is needed for use in other species with this work’s approach–so long as a multielectrode probe is implantable. In this manuscript we demon- strate electrolytic lesioning spanning two multielectrode probes across rabbits, pigs, sheep, and rhesus macaques, and our same device could be easily used with other smaller species, like rats, in which multielectrode probes have been successfully implanted [6]. Further, the approach in Khateeb, et al., is limited to superficial brain structures, due to the need for opti- cal accessibility. As noted, fiber optics could allow access to deeper structures, which would bring associated additional tissue damage, but deeper structure lesioning was not demon- strated. In contrast, the approach presented here can be used in any region of cortex in which a multielectrode probe can be implanted, which, depending on the probe used, does not limit it to surface structures. For example, we demonstrated use of our lesioning tech- nique with a linear U-probe (Fig. 8figure.caption.25), which could be used to reach deeper layers of cortex or specific deep cortical structures. In both techniques, the location of the lesion is tied to the location of the electrophysiology (for Khateeb et al., wherever the cra- nial window and ECoG array are; for this technique, wherever the multielectrode probe has been implanted), which ensures that the electrophysiology will include recordings from the perilesional area. Neither work addresses the potential of their technique to induce chronic post-lesion behavioral effects, which is a key goal for future work.”

There is a lack of quantitative histological analysis of the change in neuronal morphology and loss.

We appreciate the reviewer’s desire for a quantitative histological analysis, however this falls out- side of the scope of this manuscript. We are not attempting to make strong claims about the number of neurons lost through lesioning or thoroughly characterize morphological changes in the neurons. The histology is intended to show that lesioning did lead to a loss of neurons, but the precise num- ber of neurons lost is neither in scope nor is likely to be highly conserved across lesions.

There is a lack of histology data across animals and on the reliability of their lesioning techniques across animals and experiments.

We thank the reviewer for this point. As stated above, we have now added Fig. 4 and Fig. 5, which includes volume estimates based on the histology from more of our ex vivo and in vivo testing across animals.

There is a lack of data on changes in cortical layers and structures across the lesioning and non- lesioning electrodes.

We acknowledge that the histology does not have the level of detail that is expected from many modern studies. However, the goal here was dramatically different: we sought to calibrate a novel lesion device, ensure it’s safe use in large mammals (specifically, non-human primates) and pro- vide estimates of the lesion size to compare with the literature. The extent of histology that could be performed and the tools available to us prevent such an in depth analysis. We can say based on shank length of the Utah arrays used and known anatomy that we have affected layer 2/3 and maybe a bit of layer 4.

Reviewer 1 (Recommendations For The Authors):

Figure 5b. It would be helpful if the author could plot the delta match separately for the lesion elec- trodes, near neighbor electrodes, and far neighbors. This would help understand the lesion effect, specifically whether the effect is selective (e.g., more potent for the lesion and adjacent electrodes.)

The fact that neuron loss is not particularly selective can already be seen in the spike waveform plots, arranged spatially on the array. Plenty of clear change is observed far from the lesion elec- trodes (marked with black dots) as well as nearby. We have made mention of this localized non- specificity in the main text and have ensured to remphasize in the figure legened. While a nice suggestion, we currently don’t feel this result rises to the level of a figure given it is not highly specific spatially.

Reviewer 2 (Recommendations For The Authors):

Overall the quality of the paper, the figures and the analysis used could be significantly improved. There is a lack of scientific rigor in the presentation of figures and analysis techniques. It is not clear what the authors are trying to communicate through the figures and their choice of figures to show is confusing (see below).

We thank the reviewer for their pointed critiques and believe we have addressed their concerns with many changes to the text, a revamped waveforms analysis, and both the expansion and addition of results.

The neurophysiology data shown doesn’t suggest neuronal loss, it only shows change which needs strong control data to show it is due to a lesion.

As detailed below, we have presented a revised analysis that provides this control. While the reviewer is right to point out we can distinguish actual neuron loss from neuron silencing, we be- lieve the new analysis rigorously indicates new rates of sample turnover beyond those expected from healthy state.

The histology figure should be replaced with a high-quality representation without folds.

We understand the reviewer’s suggestion. While ideally we would have many histology slices from each lesion, due to cost, we were only able to collect one histology slice per lesion. The folds were introduced by the company that performed the H&E staining, and we unfortunately cannot remove the folds. Therefore, despite the folds, this is the best and only image from this lesion. We hope that the markings on the figure and the comment in the caption is sufficient to explain to readers that the folds are not a result of the lesion but instead a result of the histology process.

The authors suggest that this lesioning method will be compatible with any available multielec- trode probe theoretically. Since all testing was done with a Utah array, it will be helpful to add an explanation about potential constraints that will make a given array compatible with this method.

We thank the reviewer for this suggestion. As stated above, we have both added a demonstration of use in a different multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

The authors should cite and discuss previous studies using electrolytic lesioning in awake-behaving animals to study the causal connection between the brain and behavior. (One example study: Morissette MC, Boye SM. Electrolytic lesions of the habenula attenuate brain stimulation reward. Behavioural brain research. 2008 Feb 11;187(1):17-26.)

We thank the reviewers for this suggestion. We have added a mention of existing electrolytic le- sioning studies on Page 2, Line 88.

“Prior termination studies mostly measure behavioral output, with no simultaneous measures of neuronal activity during the behavior, impairing their ability to provide insight into the causal connection between the brain and behavior [7]–[11], or with no baseline (i.e., pre- lesion) measures of neuronal activity [4].”

The authors should compare their technique with other recent lesioning studies in primates (e.g. Khateeb et al, 2022)

We again thank the reviewer for this point. Specifically not mentioning Khateeb et al. 2022 was a submission error on our part; we cited the paper in Appendix 2 in the version uploaded to the eLife submission portal, but we had uploaded the version prior to citing it to bioRxiv. We have combined addressing this with addressing a previous comment, as mentioned above, with a section in the Discussion on Page 16, Line 434.

In Appendix 2, the authors suggest that a major limitation of optogenetics and chemogenetic in- activation methods is the lack of rhesus-compatible constructs. However, several viral constructs have successful implementation in rhesus monkeys so far (e.g. Galvan A, Stauffer WR, Acker L, El-Shamayleh Y, Inoue KI, Ohayon S, Schmid MC. Nonhuman primate optogenetics: recent advances and future directions. Journal of Neuroscience. 2017 Nov 8;37(45):10894-903; Tremblay et al, Neuron 2020)

We thank the reviewer for pointing us to these papers. We have added a more thorough description of what we meant by lack of rhesus-compatible constructs in that Appendix.

“However, other challenges exist with using optogenetics as an inactivation method in nonhu- man primates, including difficulty reliably affecting behavior [12]. While several constructs for rhesus macaques have been developed [13], [14], reports of successfully inducing be- havioral effects have a small effect size and are less numerous than might be expected [12], and several null results have been published [15]–[17]. Other remaining challenges include the need to develop a head-mounted, battery powered light delivery system for multi-day delivery of light and difficulty integrating illumination with simultaneous chronic neuro- electrophysiology.”

For Figure 5b, only pairwise comparison results from monkey U (L11-14) are shown. It is unclear why such results from monkey H were shown in Figure 5a but not in 5b.

We thank the reviewer for pointing out this unconventional one monkey result. As described in the original submission, we previously omitted Monkey H from the analysis in Figure 5b (now Figure 7) since some of the lesions were closely spaced together, preventing well defined pre- and post- lesion rates of turnover. Never-the-less we have included Monkey H in all the revised analysis and believe even the less cleanly separated data shows useful indications of neuron loss or silencing evoked by the lesion.

Behavioral data (during a motor task) from the awake behaving monkeys (U and H) would greatly strengthen the claim that this lesioning method is capable of creating a behavioral effect and can be adopted to study the relationship between neural function and behavior outcomes.

While we are grateful for the reviewer’s interest in the application of our lesioning technique to studies involving behavior, a behavioral analysis of the effects of our electrolytic lesions falls be- yond the scope of this Tools and Resources manuscript. We would also like to point out that we do not claim that we have achieved a behavioral deficit in this manuscript.

Figure 2 would benefit from an illustration of the Utah array placement and the location of the sites used for lesioning. The authors can either overlay the illustrations on the current ex-vivo and histology images or create a separate schematic to demonstrate that for the readers. Also, Figure 2B needs to be replaced with one without the folds to avoid confusion for the readers.

We have added Figure 2 - figure supplement 1, which shows both the location within the Utah array of the two electrodes used to create the lesions as well as the relative size of the surface area of the lesion and the array. Unfortunately, as the lesion was created under the array, the exact location of the array relative to the lesion is unknown.

As mentioned above, Figure 2B is the only histological image from that lesion. We hope that the markings in the image as well as the caption sufficiently explain that the folds are unrelated to the lesion itself.

Figure 3, the conical region is not well delineated. Data across animals and lesion volume with respect to different parameters should be included.

We have included a supplemental figure, Figure 3 - figure supplement 1, where we have used a dashed white line to clearly indicate the area of damaged parenchyma, in case it was not clear in Figure 3a. We have also added volume estimates from lesions across animals and different param- eters. The ex vivo estimates are shown in Figure 4 and the in vivo estimates are shown in Figure 5.

Figure 4: it is not clear what is being communicated, and where the voltage traces are from.

We thank the reviewer for noting this confusion. We have added some lines in the text to explain what the voltage traces show, both in the caption to Fig. 6 and in the text on Page 7, Line 238.

“Traces only capture the values while the lesioning device was turned on (45 seconds for most lesions and 50 seconds for lesion H200120). A) Voltage traces. Discontinuity at the beginning of the traces indicates transient voltages that were too rapid to be captured by the voltmeter, lasting between 0.13 and 0.33 s. The fluctuating voltages, especially the rapid in- crease in voltage at the beginning of lesioning, emphasize the importance of using a current source to deliver consistent amounts of current into the brain.”

“The voltage across the microelectrode array fluctuated much more than the current did, em- phasizing that we made the correct choice in using a current source to ensure delivery of consistent amounts of current into the brain (Fig. 6figure.caption.19).”

Figure 5: why did the authors choose to use matching units as a measure of the lesion? It is surprising that there are still units on the location that the authors claim to be a lesion. To clarify that it would be helpful to show the location of the lesion in Figure 4a. Also, what can we conclude about the lesion induction when we see units on the lesion electrode? The change in unit match shows that there is a change in the network (although the authors need to show control for that so we know those changes don’t happen due to natural dynamics). It is not clear what is the time duration for pre-pre and post-post (i.e. minutes, seconds, hours). Do these comparisons come from the same time frame or are they coming from two fragments of time for both pre and post- conditions?

Aside from post-mortem histology and tissue assays, there is no good way to confirm neuron loss with chronically implanted electrode arrays in nonhuman primates. Waveforms were chosen as they are the one readily isolated physical measure of the system we are injuring. Although functional measures of activity could indicate neuron loss (topic of following papers), there are many conceivable changes in firing rate patterns that could manifest spuriously as loss, making the estimation of loss even more ambiguous and challenging this way.

We believe the new Figure 7 will make the procedure much more clear, while also providing the control requested by the reviewer, illustrating that new statistical categories of altered waveforms emerge during a lesion, beyond those associated with typical changes in waveform composition within multi-unit recordings seen during recording sample turnover fom healthy animals. We further note that by confining this analysis to four day spans at most, we have limited the impact of daily sample turnover described in the literature (Gallego, 2020).

The time duration for pre-session versus pre-session (pre-post and post-post), is some multiple of the approximate 24 hours between each daily recording session. Therefore, since restricting our- selves to four days separation, between 24 and 96 hours. Spikes are sampled from successful trial periods (so on the order of seconds, compiled into minutes across the whole recording session). Although already described in the main text, these points have been reemphasized in the figure legend.

CNO (line 931) needs to be explained.

We thank the reviewer for this point. We have defined CNO and its relevance in Appendix 2.

“Additionally, chronic inactivation over days may be logistically challenging, as the half life of clozapine N-oxide (CNO, a ligand used to activate DREADD receptors) is on the order of hours.”

eLife assessment

This paper reports a valuable new method for creating localized damage to candidate brain regions for functional and behavioral studies. The authors present solid support for their ability to create long-term local lesions with mm spatial resolution. The paper is likely to be of broad interest to brain researchers working to establish causal links between neural circuits and behavior.

Reviewer #1 (Public Review):

In the paper, the authors illustrated a novel method for Electrolytic Lesioning through a microelectronics array. This novel lesioning technique is able to perform long-term micro-scale local lesions with a fine spatial resolution (mm). In addition, it allows a direct comparison of population neural activity patterns before and after the lesions using electrophysiology. This new technique addresses a recent challenge in the field and provides a precious opportunity to study the natural reorganization/recovery at the neuronal population level after long-term lesions. It will help discover new causal insights investigating the neural circuits controlling behavior.

Comments on revised version:

We appreciate the revisions made by the authors in response to our comments on the previous version of their manuscript. They carefully addressed the majority of the concerns and performed additional experiments. The new figure illustrating the lesion volume as a function of electrolytic lesioning parameters provides a valuable reference for future experiments. In addition, the latest results on different versions of passive multielectrode probes, U-probe, demonstrate that the technique is applicable beyond the specific technical setup they employ. Overall, we believe that the revised manuscript is significantly improved.

Reviewer #2 (Public Review):

This work by Bray et al. presented a customized way to induce small electrolytic lesions in the brain using chronically implanted intracortical multielectrode arrays. This type of lesioning technique has the benefit of high spatial precision and low surgical complexity while allowing simultaneous electrophysiology recording before, during, and after the lesion induction. The authors have validated this lesioning method with a Utah array, both ex vivo and in vivo using pig models and awake-behaving rhesus macaques. Given its precision in controlling the lesion size, location, and compatibility with multiple animal models and cortical areas, the authors believe this method can be used to study cortical circuits in the presence of targeted neuronal inactivation or injury and to establish causal relationships before behavior and cortical activity.

Strengths:

- Overall the techniques, parameters, and data analysis methods are better described in the revised version.

- The authors added the section "Relationship Between Applied Current and Lesion Volume" as well as Figure 4 and 5 to address our comments regarding parameter testing. Multiple combinations of current amplitude and duration were tested and the induced lesion volumes were estimated, providing a better picture of why certain parameters were chosen for in vivo studies.

- The authors added Figure 7 which addressed our comment "more evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning." They went into more details to explain the observed changes in pairwise comparisons of spike waveforms (difference in projected radii). Particularly in Fig 7C, they identified a new cluster from the pre-post lesioning group, which effectively represented neuronal loss from the<br /> recorded population.

- The authors discussed their method in the context of other literature and stating its strength and limitation.

Major comments:

-The lack of histology limits the validation of lesion induction, ideally cell loss and neuronal loss in vivo needs to be quantified. In addition based on the lack of access to histology, it is not clear how the lesion volumes are calculated which also impacts the scientific rigor of the work. The authors mention that layers 2/3 and maybe 4 have been impacted. The lack of information on the extent of the lesion severely limits the use of their technique for neuroscience experiments.

-The lack of histology in combination with behavioral measures still limits the impact of the paper in the context of NHP research.

- Figure 5 involves fitting an exponential model to the generated lesion volume given the applied current amplitude and duration. However, the data from ex vivo sheep and pig cortex with the same current amplitude & three durations showed very large variability in lesion volume at Time = 2min (larger than the difference from 2 to ~2.2min). Very limited data points exist for the other two parameter combinations. These may suggest that the exponential fit is not the best model in this scenario.

- Regarding the comment on neuronal inactivation, the authors still did not show any evidence of single unit activity loss or changes in local field potential/multi-unit activity from the region being lesioned.

- Regarding this comment "The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study" the authors explained that "a lesion was performed on a sedated rhesus macaque (monkey F) who was subsequently euthanized due to unrelated health complications, in order to further verify safety before use in awake-behaving rhesus" but still no histology data is shown regarding monkey F to demonstrate this verification. Given that NHPs are highly valuable resources, it's important to make use of all collected data and to show that the induced lesion is comparable to those in the pig cortex.

Reviewer #1 (Public Review):

This paper describes "Ais", a new software tool for machine-learning-based segmentation and particle picking of electron tomograms. The software can visualise tomograms as slices and allows manual annotation for the training of a provided set of various types of neural networks. New networks can be added, provided they adhere to a Python file with an (undescribed) format. Once networks have been trained on manually annotated tomograms, they can be used to segment new tomograms within the same software. The authors also set up an online repository to which users can upload their models, so they might be re-used by others with similar needs. By logically combining the results from different types of segmentations, they further improve the detection of distinct features. The authors demonstrate the usefulness of their software on various data sets. Thus, the software appears to be a valuable tool for the cryo-ET community that will lower the boundaries of using a variety of machine-learning methods to help interpret tomograms.

eLife assessment

This work describes a new software platform for machine-learning-based segmentation of and particle-picking in cryo-electron tomograms. The program and its corresponding online database of trained models will allow experimentalists to conveniently test different models and share their results with others. The paper provides solid evidence that the software will be valuable to the community.

Reviewer #2 (Public Review):

Summary:

Last et al. present Ais, a new deep learning-based software package for the segmentation of cryo-electron tomography data sets. The distinguishing factor of this package is its orientation to the joint use of different models, rather than the implementation of a given approach. Notably, the software is supported by an online repository of segmentation models, open to contributions from the community.

The usefulness of handling different models in one single environment is showcased with a comparative study on how different models perform on a given data set; then with an explanation of how the results of several models can be manually merged by the interactive tools inside Ais.

The manuscripts present two applications of Ais on real data sets; one is oriented to showcase its particle-picking capacities on a study previously completed by the authors; the second one refers to a complex segmentation problem on two different data sets (representing different geometries as bacterial cilia and mitochondria in a mouse neuron), both from public databases.

The software described in the paper is compactly documented on its website, additionally providing links to some YouTube videos (less than an hour in total) where the authors videocapture and comment on major workflows.

In short, the manuscript describes a valuable resource for the community of tomography practitioners.

Strengths:

A public repository of segmentation models; easiness of working with several models and comparing/merging the results.

Weaknesses:

A certain lack of concretion when describing the overall features of the software that differentiate it from others.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Last and colleagues describe Ais, an open-source software package for the semi-automated segmentation of cryo-electron tomography (cryo-ET) maps. Specifically, Ais provides a graphical user interface (GUI) for the manual segmentation and annotation of specific features of interest. These manual annotations are then used as input ground-truth data for training a convolutional neural network (CNN) model, which can then be used for automatic segmentation. Ais provides the option of several CNNs so that users can compare their performance on their structures of interest in order to determine the CNN that best suits their needs. Additionally, pre-trained models can be uploaded and shared to an online database.

Algorithms are also provided to characterize "model interactions" which allows users to define heuristic rules on how the different segmentations interact. For instance, a membrane-adjacent protein can have rules where it must colocalize a certain distance away from a membrane segmentation. Such rules can help reduce false positives; as in the case above, false negatives predicted away from membranes are eliminated.

The authors then show how Ais can be used for particle picking and subsequent subtomogram averaging and for the segmentation of cellular tomograms for visual analysis. For subtomogram averaging, they used a previously published dataset and compared the averages of their automated picking with the published manual picking. Analysis of cellular tomogram segmentation was primarily visual.

Strengths:

CNN-based segmentation of cryo-ET data is a rapidly developing area of research, as it promises substantially faster results than manual segmentation as well as the possibility for higher accuracy. However, this field is still very much in the development and the overall performance of these approaches, even across different algorithms, still leaves much to be desired. In this context, I think Ais is an interesting package, as it aims to provide both new and experienced users with streamlined approaches for manual annotation, access to a number of CNNs, and methods to refine the outputs of CNN models against each other. I think this can be quite useful for users, particularly as these methods develop.

Weaknesses:

Whilst overall I am enthusiastic about this manuscript, I still have a number of comments:

On page 5, paragraph 1, there is a discussion on human judgement of these results. I think a more detailed discussion is required here, as from looking at the figures, I don't know that I agree with the authors' statement that Pix2pix is better. I acknowledge that this is extremely subjective, which is the problem. I think that a manual segmentation should also be shown in a figure so that the reader has a better way to gauge the performance of the automated segmentation.

On page 7, the authors mention terms such as "emit" and "absorb" but never properly define them, such that I feel like I'm guessing at their meaning. Precise definitions of these terms should be provided.

For Figure 3, it's unclear if the parent models shown (particularly the carbon model) are binary or not. The figure looks to be grey values, which would imply that it's the visualization of some prediction score. If so, how is this thresholded? This can also be made clearer in the text.

Figure 3D was produced in ChimeraX using the hide dust function. I think some discussion on the nature of this "dust" is in order, e.g. how much is there and how large does it need to be to be considered dust? Given that these segmentations can be used for particle picking, this seems like it may be a major contributor to false positives.

Page 9 contains the following sentence: "After selecting these values, we then launched a batch particle picking process to determine lists of particle coordinates based on the segmented volumes." Given how important this is, I feel like this requires significant description, e.g. how are densities thresholded, how are centers determined, and what if there are overlapping segmentations?

The FSC shown in Figure S6 for the auto-picked maps is concerning. First, a horizontal line at FSC = 0 should be added. It seems that starting at a frequency of ~0.045, the FSC of the autopicked map increases above zero and stays there. Since this is not present in the FSC of the manually picked averages, this suggests the automatic approach is also finding some sort of consistent features. This needs to be discussed.

Page 11 contains the statement "the segmented volumes found no immediately apparent false positive predictions of these pores". This is quite subjective and I don't know that I agree with this assessment. Unless the authors decide to quantify this through subtomogram classification, I don't think this statement is appropriate.

In the methods, the authors note that particle picking is explained in detail in the online documentation. Given that this is a key feature of this software, such an explanation should be in the manuscript.

eLife assessment

In this study, camera trapping and species distribution models are used to show that human disturbance in mountain forests in the eastern Himalayas pushes medium-sized and large mammal species into narrower habitat space, thus increasing their co-occurrence. While the collected data provide a useful basis for further work, the study presents incomplete evidence to support the claim that increased co-occurrence may indicate positive interactions between species.

Reviewer #1 (Public Review):

Summary:

This study examines the spatial and temporal patterns of occurrence and the interspecific associations within a terrestrial mammalian community along human disturbance gradients. They conclude that human activity leads to a higher incidence of positive associations.

Strengths:

The theoretical framework of the study is brilliantly introduced. Solid data and sound methodology. This study is based on an extensive series of camera trap data. Good review of the literature on this topic.

Weaknesses:

The authors do not delve into the different types of association found in the study. A more ecological perspective explaining why certain species tend to exhibit negative associations and why others show the opposite pattern (and thus, can be used as indicator species) is missing. Also, the authors do not clearly distinguish between significant (true) non-random associations and random associations.

Anthropogenic pressures can shape species associations by increasing spatial and temporal co-occurrence, but above a certain threshold, the positive influence of human activity in terms of species associations could be reverted. This study can stimulate further work in this direction.

Reviewer #2 (Public Review):

Summary:

This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:

The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:

The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

(1) At the lower level, we would need to better understand what the individual species do and "like" (their environmental niche).

(2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the study is not set up in a way to test this directly.

The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one could then add the layer of interspecific associations.

The change in activity and space use could be analysed by looking at the activity times and spatial distribution directly. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

The per-species models are simplistic: the predictors are only linear, and there are no statistical interactions. It is unclear how spatial autocorrelations of residuals were treated, although they form the basis for the association analysis. Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. What is the advance in ecological understanding at the community level?

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

This study examines the spatial and temporal patterns of occurrence and the interspecific associations within a terrestrial mammalian community along human disturbance gradients. They conclude that human activity leads to a higher incidence of positive associations.

Strengths:

The theoretical framework of the study is brilliantly introduced. Solid data and sound methodology. This study is based on an extensive series of camera trap data. Good review of the literature on this topic.

Weaknesses:

The authors use the terms associations and interactions interchangeably.

This is not the case. In fact, we state specifically that "... interspecific associations should not be directly interpreted as a signal of biotic interactions between pairs of species…" However, co-occurrence can be an important predictor of likely interactions, such as competition and predation. We stand by our original text.

It is not clear what the authors mean by "associations". A brief clarification would be helpful.

Our specific definition of what is meant here by spatial association can be found in the Methods section. To clarify, the calculation of the index of associations is based on the covariance for the two species of the residuals (epsilon) after consideration of all species-specific response to known environmental covariates. These covariances are modelled to allow them to vary with the level of human disturbance, measured as human presence and human modification. After normalization, the final index of association is a correlation value that varies between -1 (complete disassociation) and +1 (complete positive association).

Also, the authors do not delve into the different types of association found in the study. A more ecological perspective explaining why certain species tend to exhibit negative associations and why others show the opposite pattern (and thus, can be used as indicator species) is missing.

Suggesting the ecological underpinnings of the associations observed here would mainly be speculation at this point, but the associations demonstrated in this analysis do suggest promising areas for the more detailed research suggested.

Also, the authors do not distinguish between significant (true) non-random associations and random associations. In my opinion, associations are those in which two species co-occur more or less than expected by chance. This is not well addressed in the present version of the manuscript.

Results were considered to be non-random if correlation coefficients (for spatial association) or overlap (for temporal association) fell outside of 95% Confidence Intervals. This is now stated clearly in the Methods section.  In Figure 3—figure supplement 1-3 and Figure 4—figure supplement 1-3, p<0.01 levels are also presented.

The obtained results support the conclusions of the study.

Anthropogenic pressures can shape species associations by increasing spatial and temporal co-occurrence, but above a certain threshold, the positive influence of human activity in terms of species associations could be reverted. This study can stimulate further work in this direction.

Reviewer #2 (Public Review):

Summary:

This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:

The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:

The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

(1) At the lower level, we first need to understand what the individual species do and "like" (their environmental niche). That information is not presented, and the methods suggest that the representation of each species in the JSDM is likely to be extremely poor.

The response of each species to the environmental covariates provides a window into their environmental niche, encapsulated in the beta coefficients for each environmental covariate. This information is presented in Figure 2.

(2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the model is not set up this way, and the authors thus do a lot of indirect exploration, rather than direct hypothesis testing.

Our JSDM model is set up specifically to examine the effect of human disturbance on co-occurrence, after controlling for shared responses to environmental variables.  It directly tests the first hypothesis, since, if increase in indices of human disturbance had not tended to increase the measured spatial correlations between species as detected by the model, we would have rejected our stated hypothesis that human modification of habitats results in increased positive spatial associations between species.

Even when the focus is not the individual species, but rather their association, we need to formulate what the expectation is. The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one can then add the layer of interspecific associations.

Examining each species one by one and how each one responds to human disturbance would miss the effects of any meaningful interactions between species.  The analysis presented provides a means to highlight associations that would have been overlooked.  Future research could go on to analyze the strongest associations in the community and the strongest effects of human disturbance so as to uncover the underlying interactions that give rise to them and the mechanisms of human impact.  We believe that this will prove to be a much more productive approach than trying to tackle this problem species by species and pair by pair.

The change in activity and space use can be analysed much simpler, by looking at the activity times and spatial distribution directly. It remains unclear what the contribution of the JSDM is, unless it is able to represent this activity and spatial information, and put it in a testable interaction with human disturbance.

The topic is actually rather complicated. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

Then, in the next step, one would have to formulate specific hypotheses about which species are likely to change their associations more, and which less (based e.g. on predator-prey or competitive interactions). The data and analyses presented do not answer any of these issues.

We suggest that the so-called “simpler” approach described above is anything but simple, and this is precisely what the Joint Species Distribution Model improves upon.  As pointed out in the Introduction, simply examining spatial overlap is not enough to detect a signal of meaningful biotic interaction, since overlap could be the result of similar responses to environmental variables.  With the JSDM approach, this would not be considered a positive association and would then not imply the possible existence of meaningful interaction.

Another more substantial point is that, according to my understanding of the methods, the per-species models are very inappropriate: the predictors are only linear, and there are no statistical interactions (L374). There is no conceivable species in the world whose niche would be described by such an oversimplified model.

While interaction terms can be included in the JSDM, this would considerably increase the complexity of the models.  In previous work, we have found no strong evidence for the importance of interaction terms and they do not improve the performance of the models.

We have no idea of even the most basic characteristics of the per-species models: prevalences, coefficient estimates, D2 of the model, and analysis of the temporal and spatial autocorrelation of the residuals, although they form the basis for the association analysis!

The coefficient estimates for response to environmental variables used in the JSDM are provided in Figure 2 and Figure 2—source data 1.

Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

Also, all correlations among species should be shown for the raw data and for the model residuals: how much does that actually change and can thus be explained by the niche models?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. I failed to see any advance in ecological understanding at the community level.

We agree that the community-level response to human disturbance is a complex topic, and we believe it is also a very important one.  This research and its support of the spatial compression hypothesis, while not providing definitive answers to detailed mechanisms, opens up new lines of inquiry that makes it an important advance.  For example, the strong effects of human disturbance on certain associations that were detected here could now be examined with the kind of detailed species by species and pair by pair analysis that this reviewer appears to demand.

Reviewer #1 (Recommendations For The Authors):

L27 indicates instead of "idicates".

We thank the reviewer for catching that error.

L64 I would refer to potential interactions or just associations. It is always hard to provide evidence for the existence of true interactions.

We have revised to “potential interactions” to qualify this statement.

L69 Suggestion: distort instead of upset.

We thank the reviewer for catching that error.

L70-71 Here, authors use the term associations. Please, be consistent with the terminology throughout the manuscript.

We thank the reviewer for raising this important point.  The term “co-occurrence” appears to be used inconsistently in the literature, so we have tried to refer to it only when referencing the work of us. For us, co-occurrence means “spatial overlap” without qualification as to whether it is caused by interaction or simply by similar responses to environmental factors (see Blanchet et al. 2020, Argument 1). In our view, interactions refer to biotic effects like predation, competition, commensalism, etc., while associations are the statistical footprint of these processes.   In keeping with this understanding, in Line 73, we changed "association" to the stronger word "interaction," but in Line 76, we keep the words "spatiotemporal association", which is presumed to be the result of those interactions. In Line 91, we have changed “interactions” to “associations,” as we do not believe interactions were demonstrated in that study. 

L76 "Species associations are not necessarily fixed as positive or negative..." This sentence is misleading. I would say that species associations can vary across time and space, for instance along an environmental gradient.

We thank the reviewer for pointing out the potential for confusion.  In Line 79, we have changed as suggested.

L78 "Associations between free-ranging species are especially context-dependent" Loose sentence. Please, explain a bit further.

We have changed the sentence to be more specific; ”Interactions are known to be context-dependent; for example, gradients in stress are associated with variation in the outcomes of pairwise species interactions.”

L83-85 This would be a good place to introduce the 'stress gradient' hypothesis, which has also been applied to faunal communities in a few studies. According to this hypothesis, the incidence of positive associations should increase as environmental conditions harden.

In our review of the literature, we find that the stress gradient hypothesis is somewhat controversial and does not receive strong support in vertebrates.  We have added the phrase “…the controversial stress-gradient hypothesis predicts that positive associations should increase as environmental conditions become more severe…”

L86-88 Well, overall, the number of studies examining spatiotemporal associations in vertebrates is relatively small. That is, bird associations have not received much more attention than those of mammals. I find this introductory/appealing paragraph a bit rough. I think the authors can do better and find a better justification for their work.

We thank the reviewer for the comments.  We have rewritten the paragraph extensively to make it clearer and to provide a stronger justification for the study.

L106 "[...] resulting in increased positive spatial associations between species" I'd say that habitat shrinking would increase the level of species clustering or co-occurrence, but in my opinion, not necessarily the incidence of positive associations. It is not clear to me if the authors use positive associations as a term analogous to co-occurrence.

We thank the reviewer for raising this very important distinction.  Habitat shrinking would increase levels of species co-occurrence, but this is not particularly interested.  We wanted to test whether there were effects on species interactions, as revealed by associations.  We find that the terms association and co-occurrence are used somewhat loosely in the literature and so have made some new effort to clarify and systematize this in the manuscript.  For example, there appear to be a differences in the way “co-occurrence” is used in Boron 2023 and in Blanchet 2020. We do not use the term "positive spatial association" as analogous to "spatial co-occurrence.". Spatial co-occurrence, which for us has the meaning of spatial overlap, could simply be the result of similar reactions to environmental co-variates, not reflecting any biotic interaction. Joint Species Distribution Models enable the partitioning of spatial overlap and segregation into that which can be explained by responses to known environmental factors, and that which cannot be explained and thus might be the result of biotic interactions.  It is only the latter that we are calling spatial association, which can be positive or negative.   These associations may be the statistical footprint of biotic interactions.

Results:

Difference between random and non-random association patterns. It is not clear to me if the reported associations are significant or not. The authors only report the sign of the association (either positive or negative) but do not clarify if these associations indicate that two species coexist more or less than expected by chance. In my opinion, that is the difference between true ecological associations (e.g., via facilitation or competition effects) and random co-existence patterns. This is paramount and should be addressed in a new version of the manuscript.

This information is provided in Figure 3—figure supplement 1,2,3 and Figure 4—figure supplement 1,2,3.  This is referenced in the text as follows, “… correlation coefficients for 18 species pairs were positive and had a 95 % CI that did not overlap zero, and the number increased to 65 in moderate modifications but dropped to 29 at higher modifications" and so on. This criterion for significance (ie., greater than expected by chance) is now stated at the end of the Materials and methods.  In Figure 3—figure supplement 1,2,3 and Figure 4—figure supplement 1,2,3, those correlations that were significant at p<0.01 are also shown.

I am also missing a more ecological explanation for the observed findings. For instance, the top-ranked species in terms of negative associations is the red fox, whereas the muntjac seems to be the species whose presence can be used as an indicator for that of other species. What are the mechanisms underlying these patterns? Do red foxes compete for food with other species? Do the species that show positive associations (red goral, muntjac) have traits or a diet that are more different from those of other species? More discussion on these aspects (role of traits and the trophic niche) would be necessary to better understand the obtained results.

The purpose of this paper was to test the compression hypotheses, and we have tried to keep that as the focus.  However, the analysis does open up interesting lines of inquiry for future research to decipher the details of the interactions between species and the mechanisms by which human disturbance facilitates or disrupts these interactions. The reviewer raises some interesting possibilities, but at this point, any discussion along these lines would be largely speculation and could lengthen the paper without great benefit. 

Reviewer #2 (Recommendations For The Authors):

The manuscript should be accompanied by all data and code of analysis.

All data and RScripts have been made available in Science Data Bank: https://doi.org/10.57760/sciencedb.11804.

The sentence "not much is known" is weak: it suggests the authors did not bother to quantify what IS known, and simply waved any previous knowledge aside. Surely we have some ideas about who preys on whom, and which species have overlapping resource requirements (e.g., due to jaw width). For those, we would expect a particularly strong signal, if the association is indeed indicative of interactions.

We believe that the reviewer is referring to the statement in Line 90-92 about the lack of understanding of the resilience of terrestrial mammal associations to human disturbance.  We have added a reference to one very recent publication that addresses the issue (Boron et al., 2023), but otherwise we stand by our statement. We have, however, added a qualifier to make it clear that we did indeed look for previous knowledge; "However, a review of the literature indicates that ...."

Figures:

Fig. 1. This reviewer considers that this is too trivial and should be deleted.

This is a graphical statement of the hypotheses and may be helpful to some readers.

Fig. 2. Using points with error bars hides any potential information.

Done as suggested.

That only 4 predictors are presented is unacceptably oversimplified.

Only 4 predictors are included because, in previous work, we found that adding additional predictors or interactions did little to improve the model’s performance (Li et al. 2018, 2021 and 2022) and could lead to over-fitting.

Fig. 5. and 6. aggregate extremely strongly over species; it remains unclear which species contribute to the signal, and I guess most do not.

The number of detection events presented in Table 1 should help to clarify the relative contribution of each species to the data presented in Figures 5 and 6.

This reviewer considers that the introduction 'oversells' the paper.

L55: can you give any such "unique ecological information"

L60: Lyons et al. (Kathleen is the first name) has been challenged by Telford et al. (2016 Nature) as methodologically flawed.

The first name has been deleted.  The methodological flaw has to do with interpretation of the fossil record and choice of samples, not with the need to partition shared environmental preferences and interactions.

L61 contradicts line 64: Blanchet et al. (2022, specifying some arguments from Dormann et al. 2018 GEB) correctly point out that logically one cannot infer the existence or strength from co-occurrence data. It is thus wrong to then claim (citing Boron et al.) that such data "convey key information about interactions". The latter statement is incorrect. A tree and a beetle can have extremely high association and nothing to do with each other. Association does not mean anything in itself. When two species are spatially and temporally non-overlapping, they can exhibit perfect "anti-association", yet, by the authors' own definition, cannot interact.

We believe that the reviewer’s concerns arise from a misunderstanding of how we use the term association.  In our usage, an association is not the same as co-occurrence or overlap, which may simply be the result of shared responses to environmental variables.  The co-occurring tree and beetle would not be found to have any association in our analysis, only shared environmental sensitivities.  In contrast, associations can be the statistical footprint of interactions, and would be overlaid onto any overlap due to similar responses to the environment.  In the case of negative associations, such as might be the result of competitive exclusion or avoidance of predators, the two species would share environmental responses but show lower than expected spatial overlap.  Even though they might be only rarely found in the same vicinity, they would indeed be interacting when they were together.

Joint Species Distribution Models "allow the partitioning of the observed correlation into that which can be explained by species responses to environmental factors... and that which remains unexplained after controlling for environmental effects and which may reflect biotic interactions." (Garcia Navas et al. 2021). It is the latter that we are calling “associations.”

L63: Gilbert reference: Good to have a reference for this statement.

This point is important, but the reviewer’s comments below have made it clear that it is even more important to point out that strong interactions should be expected to lead to significant associations.  We have added a statement to clarify this.

L70-72: Incorrect, interactions play a role, not associations (which are merely statistical).

In this, we agree, and we have revised the statement to refer to interactions, not associations. In our view, an interaction is a biological phenomenon, while an association is the resulting statistical signal that we can detect.

L75: Associations tell us nothing, only interactions do. Since these can not be reliably inferred, this statement and this claim are wrong.

We thank the reviewer for raising this point, but we beg to disagree. Strong interactions should be expected to lead to significant associations that can be detected in the data. Associations, which can be measured reliably, are the evidence of potential interactions, and hence associations can tell us a great deal.  We have added a note to this effect after the Gilbert reference above to clarify this point.

However, we do accept that associations must be interpreted with caution. As Blanchet et al. 2020 explain, " …the co-occurrence signals (e.g. a significant positive or negative correlation value) estimated from these models could originate from any abiotic factors that impact species differently. Therefore, this correlation cannot be systematically interpreted as a signal of biotic interactions, as it could instead express potential non-measured environmental drivers (or combinations of them) that influence species distribution and co-distribution.”  Or alternatively an association could be the result of interaction with a 3rd species. 

L87: Regarding your claim, how would you know you DO understand? For that, you need to formulate an expectation before looking at the data and then show you cannot show what you actually measure. (Jaynes called this the "mind-projection fallacy".)

We are not sure if the reviewer is criticizing our paper or the entire field of community ecology.  Perhaps it is the statement that “….resilience of interspecific spatiotemporal associations of terrestrial mammals to human activity remains poorly understood….”  Since we are confident that the reviewer believes that mammals do interact, we guess that it is the term “association” that is questioned.  We have revised this to “…the impacts of human activity on interspecific interactions of terrestrial mammals remains poorly understood…” 

In this particular case, we did formulate an expectation before looking at the data, in the form of the two formal hypotheses that are clearly stated in the Introduction and illustrated in Figure 1. If the hypotheses had not been supported, then we would have accepted that we do not understand. But as the data are consistent with the hypotheses, we submit that we do understand a bit more now.

eLife assessment

This study provides a valuable resource by thoroughly benchmarking multiple sequencing-based tRNA quantification methods. The suggested best practice is supported by solid evidence from in silico experiments in multiple scenarios. The major weakness of the manuscript is the incomplete validation of newly generated experimental datasets.

Reviewer #1 (Public Review):

Summary:

In the manuscript titled "Benchmarking tRNA-Seq quantification approaches by realistic tRNA-Seq data simulation identifies two novel approaches with higher accuracy," Tom Smith and colleagues conducted a comparative evaluation of various sequencing-based tRNA quantification methods. The inherent challenges in accurately quantifying tRNA transcriptional levels, stemming from their short sequences (70-100nt), extensive redundancy (~600 copies in human genomes with numerous isoacceptors and isodecoders), and potential for over 100 post-transcriptional chemical modifications, necessitate sophisticated approaches. Several wet-experimental methods (QuantM-tRNA, mim-tRNA, YAMAT, DM-tRNA, and ALL-tRNA) combined with bioinformatics tools (bowtie2-based, SHRiMP, and mimseq) have been proposed for this purpose. However, their practical strengths and weaknesses have not been comprehensively explored to date. In this study, the authors systematically assessed and compared these methods, considering factors such as incorrect alignments, multiple alignments, misincorporated bases (experimental errors), truncated reads, and correct assignments. Additionally, the authors introduced their own bioinformatic approaches (referred to as Decision and Salmon), which, while not without flaws (as perfection is unattainable), exhibit significant improvements over existing methods.

Strengths:

The manuscript meticulously compares tRNA quantification methods, offering a comprehensive exploration of each method's relative performance using standardized evaluation criteria. Recognizing the absence of "ground-truth" data, the authors generated in silico datasets mirroring common error profiles observed in real tRNA-seq data. Through the utilization of these datasets, the authors gained insights into prevalent sources of tRNA read misalignment and their implications for accurate quantification. Lastly, the authors proposed their downstream analysis pipelines (Salmon and Decision), enhancing the manuscript's utility.

Weaknesses:

As discussed in the manuscript, the error profiles derived from real-world tRNA-seq datasets may still harbor biases, as reads that failed to "align" in the analysis pipelines were not considered. Additionally, the authors did not validate the efficacy of their "best practice" pipelines on new real-world datasets, preferably those generated by the authors themselves. Such validation would not only confirm the improvements but also demonstrate how these pipelines could alter biological interpretations.<br /> Because tRNA-sequencing methods have not been widely used (compared to mRNA-seq), many readers would not be familiar with the characteristics of different methods introduced in this study (QuantM-tRNA, mim-tRNA, YAMAT, DM-tRNA, and ALL-tRNA; bowtie2-based, SHRiMP, and mimseq; what are the main features of "Salmon?"). The manuscript will read better when the basic features of these methods are described in the manuscript, however brief.

Reviewer #2 (Public Review):

Summary:

The authors provided benchmarking study results on tRNA-seq in terms of read alignment and quantification software with optimal parameterization. This result can be a useful guideline for choosing optimal parameters for tRNA-seq read alignment and quantification.

Strengths:

Benchmarking results for read alignment can be a useful guideline to choose optimal parameters and mapping strategy (mapping to amino acid) for various tRNAseq.

Weaknesses:

The topic is highly specific, and the novelty of the analysis might not be widely useful for general readers.

Some details of the sequencing data analysis pipeline are not clear for general readers:

(1) The explanation of the parameter D for bowtie2 sounds ambiguous. "How much effort to expend" needs to be explained in more detail.

(2) Please provide optimal parameters (L and D) for tRNA-seq alignment.

(3) I think the authors chose L=10 and D=100 based on Figure 1A. Which dataset did you choose for this parameterization among ALL-tRNAseq, DM-tRNAseq, mim-tRNAseq, QuantM-tRNA-seq, and YAMAT-seq?

(4) Salmon does not need a read alignment process such as Bowtie2. Hence, it is not clear "Only results from alignment with bowtie2" in Figure legend for Figure 4a.

Author response:

We thank the reviewers for their critical appraisal of our manuscript. We will address the points of confusion and/or lack of clarity in a revised manuscript. We agree with reviewer 1 that applying the best practice pipeline(s) on new experimental data and comparing this approach with current practices would be a useful demonstration of how this alters the biological interpretation. This is something we are in the process of completing but believe this is best addressed in a separate manuscript where we can focus on the associated biological findings, allowing this manuscript to remain focused on the accurate quantification of tRNA-Seq data.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

This manuscript provides useful information about the lipid metabolite 15d-PGJ2 as a potential regulator of myoblast senescence. The authors provide experimental evidence that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas. However, the manuscript is incomplete in its current form, as it lacks robust support from the data regarding the main conclusions related to senescence and technical concerns related to the senescence models used in this study.

We are grateful to the editors and the reviewers for their time and comments in sharpening the science and the writing of the manuscript. We have attached a detailed response to emphasize that the manuscript does include robust evidence regarding the claims, which could have been missed during the review process. We have provided a better context for these points now.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors show that upon treatment with Doxorubicin (Doxo), there is an increase in senescence and inflammatory markers in the muscles. They also show these genes get upregulated in C2C12 myoblasts when treated with conditioned media or 15d-PGJ2. 15dPGJ2 induces cell death in the myoblasts, decreases proliferation (measured by cell numbers), and decreases differentiation and fusion. 15d-PGJ2 modified Cys184 of HRas, which is required for its activation as indicated by the FRET analysis with RAF RBD. They also showed that 15d-PGJ2 activates ERK signaling, but not Akt signaling, through the electrophilic center. 15d-PGJ2 inhibits Golgi localization of HRAS (only WT, not C181 or C184 mutant). They also showed that expressing the WT HRas followed by 15d-PGJ2 treatment led to a decrease in the levels of MHC mRNA and protein, and this defect is dependent on C184. This is a well-written manuscript with interesting insights into the mechanism of action of 15d-PGJ2. However, some clarification and experiments will help the paper advance the field significantly.

Strengths:

The data clearly shows that 15d-PGJ2 has a negative role in the myoblast cells and that it leads to modification of HRas protein. Moreover, the induction of biosynthetic enzymes in the PGD2 pathway also supports the induction of 15d-PGJ2 in Doxorubicin-treated cells. Both conditioned media experiments and the 15d-PGJ2 experiments show that 15d-PGJ2 could be the active component secreted by the senescent myoblasts.

Weaknesses:

The genes that are upregulated in the muscles upon injection with Doxo are also markers for inflammation. Since Doxo is also known to induce systemic inflammation, it is important to delineate these two effects (inflammatory cells vs senescent cells). The expression of beta Gal and other markers of senescence in the tissue sections will help to delineate these.

As pointed out Doxo induces systemic inflammation along with inducing DNA damage-mediated senescence. Therefore, along with the inflammatory markers of the SASP (CXCL1/2, TNF1α, IL6, PTGS1/2, PTGDS) we also observed an increase in the mRNA levels of canonical markers of DNA damage-mediated senescence. We observed an increase in the mRNA levels of cell cycle and senescence associated proteins p16 and p21 (Fig. 1C). We also observed an increased nuclear accumulation of p21 (Fig. 1A) and increased levels of phosphorylated H2A.X in the nucleus (Fig. 1B).

In Figure 2, where the defect in the differentiation of myoblasts upon treatment with 15d-PGJ2 is shown, most of the cells die within 48 hours at higher concentrations, making it difficult to perform the experiments. This also shows that 15d-PGJ2 was toxic to these cells. Lower concentrations show a decrease in the differentiation based on the lower number of nuclei in fibers and low expression of MyoD, MyoG, and MHC. However, it is unclear if this is due to increased cell death or defective differentiation. It would be a lot more informative if the cell count, cell division, and cell death could be plotted for these concentrations of the drug during the experiment.

We measured the viability of C2C12 cells after 24 hours of treatment with 15d-PGJ2 using the MTT assay and observed that the viability of cells was decreased after treatment with 15d-PGJ2 (10 µM) but not with 15d-PGJ2 (1 µM, 2 µM, 4 µM, or 5 µM) (see Fig. S2A of the updated manuscript). The results and figures of the manuscript have been updated accordingly.

Also, in the myoblast experiments, are the effects of treatment with Dox reversible?

The treatment with Doxorubicin is irreversible as the senescent phenotype was not reversed after withdrawal of Doxorubicin, even after 20 days.

In Figure 3, most of the experiments are done at a high concentration, which induces almost complete cell death within 48 hours.

Figure 3 is an acute experiment for only 1 hour, at which time no cell death was observed. Specifically, we measured the phosphorylation of Erk and Akt proteins after 1 hour of treatment with 15d-PGJ2 (10 µM) during which we did not observe any cell death. 

Even at such a high concentration of 15dPGJ2, the increase in ERK phosphorylation is minimal.

We observe a ~30% increase in the phosphorylation of Erk proteins after treatment with 15d-PGJ2 in 0.2% serum medium compared to treatment with vehicle (DMSO). This is reproducible and significant.

The experiment Figure 4C shows that C181 and C84 mutants of the HRas show higher levels in Golgi compared with WT. However, this could very well be due to the defect in palmitoylation rather than the modification with 15d-PGJ2.

Our data does not suggest higher levels of C184S mutant in the Golgi compared with WT (Fig. S4A). We observed that the ratio of HRas levels in the Golgi to the HRas levels in the plasma membrane were similar in C2C12 cells expressing HRas C184S and HRas WT (Fig. S4A graph columns 1 and 5).

Though the authors allude to the possibility that intracellular redistribution of HRas by 15d-PGJ2 requires C181 palmitoylation, the direct influence of C184 modification on C181 palmitoylation is not shown. To have a meaningful conclusion, the authors need to compare the palmitoylation and modification with 15d-PGJ2.

Palmitoylation of HRas C181S is required for the localization of HRas at the plasma membrane. The inhibition of palmitoylation of C181, either by mutation (C181S) or treatment with protein palmitoyl transferase inhibitor (2-Bromopalmitate), results in the accumulation of HRas at Golgi(Rocks et al., 2005) (Fig. S4A). Modification of HRas at C184 by 15d-PGJ2 (Fig. 3A) could inhibit the palmitoylation of HRas at C181. However, our data does not support this hypothesis as modification of HRas WT by 15d-PGJ2 does not increase the level of HRas at the Golgi, like in the case of inhibition of cysteine palmitoylation due to C181S mutation.

To test if the inhibition of myoblast differentiation depends on HRas, they overexpressed the HRas and mutants in the C2C12 lines. However, this experiment does not take the endogenous HRAs into consideration, especially when interpreting the C184 mutant. An appropriate experiment to test this would be to knock down or knock out HRas (or make knock-in mutations of C184) and show that the effect of 15d-PGJ2 disappears. 

Endogenous HRas (wild type) is present in the C2C12 cells overexpressing the EGFP-tagged HRas constructs. Therefore, we only observe a partial rescue in the differentiation after 15d-PGJ2 treatment in C2C12 cells expressing the C184S mutant (Fig. 4D and E). However, since HRas is expressed under high expression CMV promoter and in the absence of other regulatory elements, the overexpressed constructs do show a dominant effect over the endogenous HRas, showing cysteine mutant dependent inhibition of differentiation of myoblasts after treatment with 15dPGJ2 (Fig. 4D and E).

Moreover, in this specific experiment, it is difficult to interpret without a control with no HRas construct and another without the 15d-PGJ2 treatment.

The mRNA levels of MyoD, MyoG, and MHC in C2C12 cells expressing HRas constructs after treatment with 15d-PGJ2 were normalized to the mRNA levels in C2C12 cells expressing corresponding constructs and were treated with vehicle (DMSO). mRNA levels in C2C12 cells treated with vehicle were not shown as they were normalized to 1. MHC protein levels in C2C12 cells expressing HRas constructs after 15d-PGJ2 treatment were normalized to that in C2C12 cells treated with vehicle (DMSO). Since the hypothesis to study the effect of HRas cysteine mutations on the differentiation of myoblasts after treatment with 15d-PGJ2, C2C12 cells expressing HRas WT serve as adequate control. Fig. 2 shows the effect of 15dPGJ2 on muscle differentiation when HRas was not overexpressed.

Moreover, the overall study does not delineate the toxic effects of 15d-PGJ2 from its effect on the differentiation.

The inhibition of differentiation in C212 cells after treatment with 15d-PGJ2 cannot be attributed to the general toxicity of 15d-PGJ2 in cells. We show that the inhibition of differentiation of myoblasts after 15d-PGJ2 depends on modification of HRas at C184 i.e. failure to modify HRas at C184 (Fig. 3A) and resultant activation (Fig. 3B) by 15d-PGJ2 rescues this inhibition of differentiation of C2C12 cells (Fig. 4D and E), dissecting the inhibition of differentiation of myoblasts by 15d-PGJ2 from general toxic effects of 15d-PGJ2 on cell physiology.

Please note that the effect of 15d-PGJ2 on cell physiology is context-specific. On one hand, 15d-PGJ2 has been shown to exert tumor-suppressor effects by inhibiting the proliferation of ovarian cancer cells and lung adenocarcinoma cells (de Jong et al., 2011; Slanovc et al., 2024), 15d-PGJ2 also exerts pro-carcinogenic effects by induction of epithelial to mesenchymal transition in breast cancer cells MCF7 and inhibition of tumor-suppressor protein p53 in MCF7 and PC-3 cells (Choi et al., 2020; Kim et al., 2010).

Reviewer #2 (Public Review):

Summary:

In this study, Swarang and colleagues identified the lipid metabolite 15d-PGJ2 as a potential component of senescent myoblasts. They proposed that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas, suggesting its potential as a target for restoring muscle homeostasis post-chemotherapy.

Strengths:

The regulation of HRas by 15d-PGJ2 is well controlled.

Weaknesses:

The novelty of the study is compromised as the activation of PGD and 15d-PGJ2, as well as the regulation of HRas and cell proliferation, have been previously reported. 

Literature does not support this statement, and it is important to clarify this misimpression for the field as a whole. 

Let us clarify- 

Covalent modification of HRas by 15d-PGJ2 has been reported only twice in the literature(Luis Oliva et al., 2003; Yamamoto et al., 2011) in fibroblasts and neurons respectively. 

Interaction between Hras and 15d-PGJ2 in skeletal muscles has not been shown before, even though both Hras and 15d-PGJ2 are shown to be key regulators of muscle homeostasis. 

Activation of Hras by 15d-PGJ2 was reported first by Luis Oliva et al (Luis Oliva et al., 2003). However, this study does not comment on the functional implications of activation of Hras signaling. 

Recently, our lab contributed to a study where the functional implication of activation of Hras signaling due to covalent modification by 15d-PGJ2 was shown in the maintenance of senescence phenotype (Wiley et al., 2021). 

15d-PGJ2 was shown to inhibit the differentiation of myoblasts by Hunter et al (Hunter et al., 2001). This study hypothesized that the inhibition of myoblast differentiation is via 15d-PGJ2 mediated activation of the PPARγ signaling, the study also showed inhibition of myoblast differentiation independent of PPARγ activity, suggesting the presence of other mechanisms.

This is the first study to show a molecular mechanism where activation of Hras signaling in skeletal myoblasts due to covalent modification by 15d-PGJ2 at C184 of Hras inhibits the differentiation of skeletal myoblasts.

Additionally, there are major technical concerns related to the senescence models, limiting data interpretation regarding the relevance to senescent cells.

Major concerns:

(1) The C2C12 cell line is not an ideal model for senescence study due to its immortalized nature and lack of normal p16 expression. A more suitable myoblasts model is recommended, with a more comprehensive characterization of senescence features.

C2C12 is a good model for DNA damage-based senescence that is used in this manuscript. Several reports in the literature have shown the induction of senescence in C2C12 cells. Moiseeva et al 2023 show induction of senescence in C2C12 cells after etoposide-mediated DNA damage. Moustogiannis et al 2021 show the induction of replicative senescence in C2C12 cells. In this study, we show that C2C12 cells undergo DNA damage-mediated senescence after treatment with Doxo. We measured the induction of senescence in C2C12 cells upon DNA damage using several physiological (Nuclear Size, Cell Size, and SA β-gal) and molecular markers (mRNA levels of p21 and SASP factors (IL6 and TGFβ), protein levels of p21) of senescence (see Fig. 1 of the updated manuscript). The results and the figures in the manuscript have been updated accordingly.

(2) The source of increased PGD or its metabolites in the conditioned medium is unclear. Including other senescence models, such as replicative or oncogeneinduced senescence, would strengthen the study.

Fig. 1E shows time-dependent increase in the expression of PGD2 biosynthetic enzymes in senescent C2C12 cells. Fig. 1F shows an increase in the levels of 15dPGJ2 secreted by senescent C2C12 cells in the conditioned medium. This data shows that senescent C2C12 cells are the source of PGD and its metabolites in the conditioned medium.

Again, C2C12 is not suitable for replicative senescence due to its immortalized status.

We and others have shown that C2C12 cells undergo senescence, and this manuscript only used DNA damage induced senescence.

(3) In the in vivo part, it is unclear whether the increased expression of PTGS1, PTGS2, and PTGDS is due to senescence or other side effects of DOXO.

We concur that this is a limitation of this study and the subsequent work will demonstrate the origin of prostaglandin biosynthesis after treatment with Doxo in vivo.

(4) Figure 2A lacks an important control from non-senescent cells during the measurement of C2C12 differentiation in the presence of a conditioned medium.

Figure 2A tests the effect of prostaglandin PGD2 and its metabolites secreted by the senescent cells on the differentiation of myoblasts. Therefore, we inhibited the synthesis of PGD2 in senescent cells by treatment with AT-56, and then collected the conditioned medium. Conditioned medium collected from senescent C2C12 cells treated with vehicle (DMSO) served as a control for the experiment, whereas differentiation of C2C12 cells without any treatment serves as a positive control.

There is no explanation of how differentiation was quantified or how the fusion index was calculated.

The fusion index was calculated using a published myotube analyzer software (Noë et al., 2022). Appropriate information has been added to the materials and methods section of the manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Line 3: Expand SA in "SA β-gal".

The manuscript has been updated accordingly (See line 3).

Line 68: HRas is highly regulated by lipid modifications.

The manuscript has been updated accordingly (See line 67).

Figures

Figure S1A seemed incomplete (maybe some processing issue).

The Figure has been updated in the revised manuscript (See Fig. S1A).

Figure S1B-H are mislabeled.

The figure has been updated in the revised manuscript (See Fig. S1C, D, E, and F).

Figures S1E-H are not mentioned in the manuscript.

The manuscript has been updated accordingly (See line 120).

Many supplementary figures are not cited in the article.

The manuscript has been updated accordingly. (See lines 85, 120, 123, 166, 225, 356, 364, 412, and 413)

Reviewer #2 (Recommendations For The Authors):

(1) Clarify the injection method for Doxorubicin in B6J mice on line 83 (IP or IM).

Mice were injected intraperitoneally with Doxorubicin (as mentioned in the materials and methods, see lines 83 and 794)

(2) Address missing information in figures or figure legends.

There is missing piece in Sup Fig 1A.

The figure has been updated in the revised manuscript (See Fig. S1A).

Correct labels in Sup Fig 1C and 1D.

The figure has been updated in the revised manuscript (See Fig. S1C, D, E, and F).

How would the authors explain the dramatic differences in the morphology of C2C12 cells treated with DOXO between bright field and SA-beta-gal staining images in Sup Fig 1B and 1C.

The SA β-gal image after treatment with Doxo does show a flattened cell morphology. Another field of view from the same experiment has been added in the figure to show the difference in the cell morphology more prominently in the revised manuscript (See Fig. 1H).

Provide explanations for Sup Fig 1E-1G, including the meaning of the y-axis and the blue dots and red lines.

We have provided an explanation for the multiple reaction monitoring mass spectrometry used to measure the concentration of 15d-PGJ2 in the conditioned medium in the revised manuscript (see lines 119-130 and the legends of Fig. S1C, D, and E)

(3) Please review the calculation of qPCR data in Figure 1C for correctness, ensuring reference samples with an average expression level of 1.

The data in Fig. 1C was plotted using 2-ΔCT instead of 2-ΔΔCT to show the variability in the expression of mRNAs isolated from animals treated with Saline.

(4) Please explain the calculation of 15d-PGJ2/cell concentration in Figure 1F and provide raw data for review, considering the substantial changes and small error bars. The method or result section lacks an explanation of how this calculation was performed. Additionally, there is no mention of the cell number count.

All the raw values (concentration of 15d-PGJ2 measured using mass spec and cell numbers counted at the time of collection of conditioned medium) are provided in the supplementary table 1. The standard curve to calculate the concentration of 15dPGJ2 in the conditioned medium is shown in Fig. S1F. The cell number was counted after trypsinization using a hemocytometer on the day of collection of the conditioned medium.

(5) Please clarify how cell number normalization and doubling time calculation were done in Fig 2B. Consider replacing the figure with a growth curve showing confluence on the y-axis for easier interpretation.

Cells were counted every 24 hours and the normalization was done to the number of cells counted on day 0 of the treatment (to consider attaching efficiency and other cell culture parameters). Doubling time was calculated as the reciprocal of the slope of the graph of log2(normalized cell number) vs time.

eLife assessment

This manuscript outlines an interaction between senescence-related 15d-PGJ2 and the proliferation and differentiation of myoblasts, with potential implications for muscle health. This manuscript is useful in understanding the role of lipid metabolite 15d-PGJ2 in myoblast proliferation and differentiation. However, in its current form, the manuscript is incomplete as there are several concerns in the statistical analysis, lack of clarity on the mechanistic details, and concerns about the use of an immortalized C2C12 myoblasts cell line to draw major conclusions related to senescence-associated secreted phenotype.

Reviewer #1 (Public Review):

Summary:

The authors show that upon treatment with Doxorubicin (Doxo), there is an increase in senescence and inflammatory markers in the muscles. They also show these genes get upregulated in C2C12 myoblasts when treated with conditioned media or 15d-PGJ2. 15dPGJ2 induces cell death in the myoblasts, decreases proliferation (measured by cell numbers), and decreases differentiation and fusion. 15d-PGJ2 modified Cys184 of HRas, which is required for its activation as indicated by the FRET analysis with RAF RBD. They also showed that 15d-PGJ2 activates ERK signaling, but not Akt signaling, through the electrophilic center. 15d-PGJ2 inhibits Golgi localization of HRAS (only WT, not C181 or C184 mutant). They also showed that expressing the WT HRas followed by 15d-PGJ2 treatment led to a decrease in the levels of MHC mRNA and protein, and this defect is dependent on C184. This is a well-written manuscript with interesting insights into the mechanism of action of 15d-PGJ2. However, some clarification and experiments will help the paper advance the field significantly.

Strengths:

The data clearly shows that 15d-PGJ2 has a negative role in the myoblast cells and that it leads to modification of HRas protein. Moreover, the induction of biosynthetic enzymes in the PGD2 pathway also supports the induction of 15d-PGJ2 in Doxorubicin-treated cells. Both conditioned media experiments and the 15d-PGJ2 experiments show that 15d-PGJ2 could be the active component secreted by the senescent myoblasts.

Weaknesses:

The genes that are upregulated in the muscles upon injection with Doxo are also markers for inflammation. Since Doxo is also known to induce systemic inflammation, it is important to delineate these two effects (Inflammatory cells vs senescent cells). The expression of beta Gal and other markers of senescence in the tissue sections will help to delineate these.

In Figure 2, where the defect in the differentiation of myoblasts upon treatment with 15d-PGJ2 is shown, most of the cells die within 48 hours at higher concentrations, making it difficult to perform the experiments. This also shows that 15d-PGJ2 was toxic to these cells. Lower concentrations show a decrease in the differentiation based on the lower number of nuclei in fibers and low expression of MyoD, MyoG, and MHC. However, it is unclear if this is due to increased cell death or defective differentiation. It would be a lot more informative if the cell count, cell division, and cell death could be plotted for these concentrations of the drug during the experiment. Also, in the myoblast experiments, are the effects of treatment with Dox reversible?

In Figure 3, most of the experiments are done at a high concentration, which induces almost complete cell death within 48 hours. Even at such a high concentration of 15dPGJ2, the increase in ERK phosphorylation is minimal.

The experiment Figure 4C shows that C181 and C84 mutants of the HRas show higher levels in Golgi compared with WT. However, this could very well be due to the defect in palmitoylation rather than the modification with 15d-PGJ2. Though the authors allude to the possibility that intracellular redistribution of HRas by 15d-PGJ2 requires C181 palmitoylation, the direct influence of C184 modification on C181 palmitoylation is not shown. To have a meaningful conclusion, the authors need to compare the palmitoylation and modification with 15d-PGJ2.

To test if the inhibition of myoblast differentiation depends on HRas, they overexpressed the HRas and mutants in the C2C12 lines. However, this experiment does not take the endogenous HRAs into consideration, especially when interpreting the C184 mutant. An appropriate experiment to test this would be to knock down or knock out HRas (or make knock-in mutations of C184) and show that the effect of 15d-PGJ2 disappears. Moreover, in this specific experiment, it is difficult to interpret without a control with no HRas construct and another without the 15d-PGJ2 treatment.

Moreover, the overall study does not delineate the toxic effects of 15d-PGJ2 from its effect on the differentiation.

Reviewer #2 (Public Review):

Summary:

In this study, Swarang and colleagues identified the lipid metabolite 15d-PGJ2 as a potential component of senescent myoblasts. They proposed that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas, suggesting its potential as a target for restoring muscle homeostasis post-chemotherapy.

Strengths:

The regulation of HRas by 15d-PGJ2 is well controlled.

Weaknesses:

(1) I still think the novelty is limited by previous published findings. The authors themselves noted that the accumulation of 15d-PGJ2 in senescent cells has been reported in various cell types, including human fibroblasts, HEPG2 hepatocellular carcinoma cells, and HUVEC endothelial cells (PMCID: PMC8501892). Although the current study observed similar activation of 15d-PGJ2 in myoblasts, it appears to be additive rather than fundamentally novel. The covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras was reported over 20 years ago (PMID: 12684535), and the biochemical principles of this interaction are likely universal across different cell types. The regulation of myogenesis by both HRas and 15d-PGJ2 has also been previously extensively reported (PMID: 2654809, 1714463, 17412879, 20109525, 11477074). The main conceptual novelty may lie in the connection between these points in myoblasts. But as discussed in another comment, the use of C2C12 cells as a model for senescence study is questionable due to the lack of the key regulator p16. The findings in C2C12 cells may not accurately represent physiological-relevant myoblasts. It is recommended that these findings be validated in primary myoblasts to strengthen the study's conclusions.

(2) The C2C12 cell line is not an ideal model for senescence study.<br /> C2C12 cells are a well-established model for studying myogenesis. However, their suitability as a model for senescence studies is questionable. C2C12 cells are immortalized and do not undergo normal senescence like primary cells as C2C12 cells are known to have a deleted p16/p19 locus, a crucial regulator of senescence (PMID: 20682446). The use of C2C12 cells in published studies does not inherently validate them as a suitable senescence model. These studies may have limitations, and the appropriateness of the C2C12 model depends on the specific research goals.<br /> In the study by Moustogiannis et al. (PMID: 33918414), they claimed to have aged C2C12 cells through multiple population doublings. However, the SA-β-gal staining in their data, which is often used to confirm senescence, showed almost fully confluent "aged" C2C12 cells. This confluent state could artificially increase SA-β-gal positivity, suggesting that these cells may not truly represent senescence. Moreover, the "aged" C2C12 cells exhibited normal proliferation, which contradicts the definition of senescence. Similar findings were reported in another study of C2C12 cells subjected to 58 population doublings (PMID: 21826704), where even at this late stage, the cells were still dividing every 2 or 3 days, similar to younger cells at early passages. More importantly, I do know how the p16 was detected in that paper since the locus was already mutated. In terms of p21, there was no difference in the proliferative C2C12 cells at day 0.<br /> In the study by Moiseeva et al. in 2023 (PMID: 36544018), C2C12 cells were used for senescence modeling for siRNA transfection. However, the most significant findings were obtained using primary satellite cells or confirmed with complementary data.<br /> In conclusion, while molecular changes observed in studies using C2C12 cells may be valid, the use of primary myoblasts is highly recommended for senescence studies due to the limitations and questionable senescence characteristics of the C2C12 cell line.

(3) Regarding source of increased PGD in the conditioned medium, I want to emphasize that it's unclear whether the PGD or its metabolites increase in response to DNA damage or the senescence state. Thus, using a different senescent model to exclude the possibility of DNA damage-induced increase will be crucial.

(4) Similarly for the in vivo Doxorubicin (Doxo) injection, both reviewers have raised concerns about the potential side effects of Doxo, including inflammation, DNA damage, and ROS generation. These effects could potentially confound the results of the study. The physiological significance of this study will heavily rely on the in vivo data. However, the in vivo senescence component is confounded by the side effects of Doxo.

(5) Figure 2A lacks an important control from non-senescent cells during the measurement of C2C12 differentiation in the presence of conditioned medium. The author took it for granted that the conditioned medium from senescent cells would inhibit myogenesis, relying on previous publications (PMID: 37468473). However, that study was conducted in the context of myotonic dystrophy type 1. To support the inhibitory effect in the current experimental settings, direct evidence is required. It would be necessary to include another control with conditioned medium from normal, proliferative C2C12 cells.

(6) Statistical analyses problems.<br /> Only t-test was used throughout the study even when there are more than two groups. Please have a statistician to evaluate the replicates and statistical analyses used.<br /> For the 15d-PGJ2/cell concentration measurements in Figure 1F, there were only two replicates, which was provided in the supplementary table after required. Was that experiment repeated with more biological replicates?<br /> For figure 1C, Fig 1F, 1G, 1J, 2C, 2E, 3A, 3E, 3F, 4D, 4E, please include each data points in bar graphs as used in Fig 1D, or at least provide how many biological replicates were used for each experiment?<br /> There is no error bar in a lot of control groups (Fig 2C, 2E, 3EF, 4E, S4B).<br /> For qPCR data in Figure 1C, the author responded in that the data in was plotted using 2-ΔCT instead of 2-ΔΔCT to show the variability in the expression of mRNAs isolated from animals treated with Saline. This statement does not align with the method section. Please revise.

(7) For Figure 1, the title may not be appropriate as there is insufficient data to support the inhibition of myoblast differentiation.

Two term that are completly related, lietrature and art, but kno we have the posibility to connect thus art to the digital world.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Eaton et al. examine the regulation of transcription directionality using a powerful genomic approach (more about the methodology below). Their data challenge the notion that the polyadenylation signal-reading Cleavage and Polyadenylation (CPA) complex is responsible for controlling promoter directionality by terminating antisense transcription. Namely, depletion of the required CPA factor RBBP6 has little effect on antisense transcription measured by POINT. They find instead that initiation is intrinsically preferential in the sense direction and additionally maintained by the activities of an alternative processing complex called Integrator, together with the kinase CDK9. In the presence of CDK9 activity, depletion of Integrator endoribonuclease INTS11 leads to globally increased transcription in the antisense direction, and minor effects in the sense direction. However, CDK9 inhibition reveals that sense transcription is also sensitive to INS11 depletion. The authors suggest that CDK9 activity is stronger in the sense direction, preventing INTS11-mediated premature termination of sense transcrpts.

Strengths:

The combination of acute depletion of the studied factors using degron approaches (important to limit possible secondary effects), together with novel and very sensitive nascent transcriptomics methods POINT and sPOINT is very powerful. The applied spike-in normalization means the analysis is more rigorous than most. Using this methodology allowed the authors to revisit the interesting question of how promoter/transcription directionality is determined.

The data quality appears very good and the fact that both global analysis as well as numerous gene-specific examples are shown makes it convincing.

The manuscript is well written and hence a pleasure to read.

We appreciate this positive assessment.

Weaknesses:

I am slightly worried about the reproducibility of the data - it is unclear to me from the manuscript if and which experiments were performed in replicate (lack of table with genomic experiments and GEO access, mentioned in more detail in below recommendations to authors), and the methods could be more detailed.

All sequencing data was deposited with GEO. Multiple biological replicates were performed for each sequencing experiment.  Bigwig files are presented as a table in the GEO submissions. This data has now been made public.

A separate discussion section would be useful, particularly since the data provided challenge some concepts in the field. How do the authors interpret U1 data from the Dreyfuss lab in light of their results? How about the known PAS-density directionality bias (more PAS present in antisense direction than in sense) - could the differential PAS density be still relevant to transcription directionality?

As suggested, we have expanded our discussion to relate our findings to existing data. We think the results from the Dreyfuss lab are very important and highlight the role of U1 snRNA in enforcing transcriptional elongation.  It does this in part by shielding PAS sequences.  Recent work from our lab also shows that U1 snRNA opposes the Restrictor complex and PNUTS, which otherwise suppress transcription (Estell et al., Mol Cell 2023).  Most recently, the Adelman lab has demonstrated that U1 snRNA generally enhances transcription elongation (Mimoso and Adelman., Mol Cell 2023).  Our work does not challenge and is not inconsistent with these studies.

The role of U1 in opposing PAS-dependent termination inspired the idea that antisense transcriptional termination may utilise PASs.  This was because such regions are rich in AAUAAA and comparatively poor in U1 binding sites. However, our RBBP6 depletion and POINT-seq data suggest that PAS-dependent termination is uncommon in the antisense direction. As such, other mechanisms suppress antisense transcription and influence promoter directionality. In our paper, we propose a major role for the Integrator complex.

We do not completely rule out antisense PAS activity and discuss the prior work that identified polyadenylated antisense transcripts. Nevertheless, this was detected by oligo-dT primed RT-PCR/Northern blotting, which cannot determine the fraction of non-polyadenylated RNA that could result from PAS-independent termination (e.g. by Integrator).  To do that requires an analysis of total nascent transcription as achieved by our POINT-seq.  Based on these experiments, Integrator depletion has a greater impact on antisense transcription than RBBP6 depletion. 

I find that the provided evidence for promoter directionality to be for the most part due to preferential initiation in the sense direction should be stressed more. This is in my eyes the strongest effect and is somehow brushed under the rug.

We agree that this is an important finding and incorporated it into the title and abstract.  As the reviewer recommends, we now highlight it further in the new discussion.

References 12-17 report an effect of Integrator on 5' of protein-coding genes, while data in Figure 2 appears contradictory. Then, experiments in Figure 4 show a global effect of INST11 depletion on promoter-proximal sense transcription. In my opinion, data from the 2.5h time-point of depletion should be shown alongside 1.5h in Figure 2 so that it is clear that the authors found an effect similar to the above references. I find the current presentation somehow misleading.

We are grateful for this suggestion and present new analyses demonstrating that our experiment in Figure 2 concurs with previous findings (Supplemental Figures 2A and B). Our original heatmap (Figure 2E) shows a very strong and general antisense effect of INTS11 loss. On the same scale, the effects in the sense direction are not as apparent, which is also the case using metaplots.  New supplemental figure 2A now shows sense transcription from this experiment in isolation and on a lower scale, demonstrating that a subset of genes shows promoter-proximal increases in transcription following INTS11 depletion.  This is smaller and less general than the antisense effect but consistent with previous findings.  Indeed, our new analysis in supplemental figure 2B shows that affected protein-coding genes are lowly expressed, in line with Hu et al., Mol Cell 2023. This explains why a sense effect is not as apparent by metaplot, for which highly expressed genes contribute the most signal.

As a result of our analyses, we are confident that the apparently larger effect at the 2.5hr timepoint (Figure 4) that we initially reported is due to experimental variability and not greater effects of extended INTS11 depletion. Overlaying the 1.5h and 2.5h datasets (Supplemental Figure 4B) revealed a similar number of affected protein-coding genes with a strong (83%) overlap between the affected genes.  To support this, we performed qPCR on four affected protein-coding transcripts which revealed no significant difference in the level of INTS11 effect after 2.5h vs 1.5h (Supplemental Figure 4C).

We now present data for merged replicates in Figures 2 and 4 which reveal very similar average profiles for -INTS11 vs +INTS11 at both timepoints. Overall, we believe that we have resolved this discrepancy by showing that it amounts to experimental variability and because the most acutely affected protein-coding genes are lowly expressed. As detailed above, we show this in multiple ways (and validate by qPCR) We have revised the text accordingly and removed our original speculation that differences reflected the timeframe of INTS11 loss.

Conclusion/assessment:

This important work substantially advances our understanding of the mechanisms governing the directionality of human promoters. The evidence supporting the claims of the authors is compelling, with among others the use of advanced nascent transcriptomics including spike-in normalization controls and acute protein depletion using degron approaches.

In my opinion, the authors' conclusions are in general well supported.

Not only the manuscript but also the data generated will be useful to the wide community of researchers studying transcriptional regulation. Also, the POINT-derived novel sPOINT method described here is very valuable and can positively impact work in the field.

We are grateful for the reviewers' positive assessment of our study.

Reviewer #2 (Public Review):

Summary:

Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper that uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:

One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full-length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

We appreciate this positive assessment.

Weaknesses:

While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figures 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

We agree that other (so far, unknown) factors promote sense transcription over antisense, which was demonstrated by our short POINT.  We have provided an expanded discussion on this in the revision. In our opinion, demonstrating that sense transcription is driven by preferential initiation in that direction is a key finding and we agree that the identification of the underlying mechanism constitutes an interesting avenue for future study.

Reviewer #3 (Public Review):

Summary:

Using a protein degradation approach, Eaton et al show that INST11 can terminate the sense and anti-sense transcription but higher activity of CDK9 in the sense direction protects it from INS11-dependent termination. They developed sPOINT-seq that detects nascent 5'-capped RNA. The technique allowed them to reveal robust transcription initiation of sense-RNA as compared to anti-sense.

Strengths:

The strength of the paper is the acute degradation of proteins, eliminating the off-target effects. Further, the paper uses elegant approaches such as POINT and sPOINT-seq to measure nascent RNA and 5'-capped short RNA. Together, the combination of these three allowed the authors to make clean interpretations of data.

We appreciate this positive assessment.

Weaknesses:

While the manuscript is well written, the details on the panel are not sufficient. The methods could be elaborated to aid understanding. Additional discussion on how the authors' findings contradict the existing model of anti-sense transcription termination should be added.

We have added more detail to the figure panels, which we hope will help readers to navigate the paper more easily. Specifically, the assay employed for each experiment is indicated in each figure panel. As requested, we provide a new and separate discussion section in the revision.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Congratulations on this important piece of work!

Some specific suggestions.

MAJOR

-The data are not available (Accession "GSE243266" is currently private and is scheduled to be released on Sep 01, 2026.) This should be corrected and as a minimum, the raw sequencing files as well as the spike-in scaled bigwig files should be provided in GEO.

We have made the data public. Raw and bigwig files are provided as part of the GEO upload.

MINOR

- It would be useful for readers if you could include catalog numbers of the reagents used in the study.

We have included this information in our revision.

- A table in experimental procedures summarizing the genomic experiments performed in this study as well as published ones reanalyzed here would be helpful.

This is now provided as part of the resources table.

- It would be easier for reviewers to evaluate the manuscript if the figure legends were included together with the figures on one page. This is now allowed by most journals.

We have used this formatting in the revision.

- Providing some captions for the results sections would be helpful.

We have included subheadings as suggested.

Reviewer #2 (Recommendations For The Authors):

Generally, I would suggest writing the experiment-type above panels where it is not immediately obvious what they are so a reader can appreciate the figures without referencing the legend. E.g. write POINT-seq on Figure 1B just to make it obvious to someone looking at the figures what methodology they are looking at. Likewise, you could write RNAPII ChIP-seq for Supplementary Figures 3D and 3E.

We have carried out this recommendation.

Can a y-axis be indicated on POINT-seq genome browser tracks? This could make them easier to interpret.

Y-axis scales are provided as RPKM as stated in the figure legends.

The authors could address/speculate in the text why there is less POINT-seq signal for the antisense transcript in the treatment condition in Figure 1B? Or could consider including a different example locus where this is not the case for clarity.

Acute depletion of poly(A) factors (like RBBP6) results in a strong read-through beyond the poly(A) signal of protein-coding genes as Figure 1 shows.  However, it also causes a reduction in transcription levels, which can be seen in the figure and is correctly noted by the reviewer in this comment.  We see this with other poly(A) factor depletions (e.g. CPSF73 and CPSF30 – Eaton et al., 2020 and Estell et al., 2021) and other labs have observed this too (e.g for CPSF73-dTAG depletion (Cugusi et al., Mol Cell 2022)).  Plausible reasons include a limited pool of free RNAPII due to impaired transcriptional termination or limited nucleotide availability due to their incorporation within long read-through transcripts. For these reasons, we have retained the example in Figure 1B as a typical representation of the effect. Moreover, the heatmap in Figure 1D fairly represents the spectrum of effects following RBBP6 loss – highlighting the strong read-through beyond poly(A) signals and the marginal antisense effects.

"The established effect of INTS11 at snRNAs was detected in our POINT-seq data and demonstrates the efficacy of this approach (Figure 2B)." The authors could explain this point more clearly in the text and describe the data - e.g. As expected, depletion of INTS11 leads to increased POINT-seq signal at the 3' end of snRNAs, consistent with defects in transcriptional termination. This is highlighted by the RNU5A-1 and RNU5B-1 loci (Figure 2B).

We agree and have added more context to clarify this.

I would suggest adjusting the scale of the heatmap in Figure 2E - I think it would be easier to interpret if the value of 0 was white - with >0 a gradient of orange and <0 a gradient of blue (as is done in Figure 1C). I think making this change would make the point as written in the text clearer i.e. "heatmap analysis demonstrates the dominant impact of INTS11 on antisense versus sense transcription at most promoters (Figure 2E)." I'm assuming most of the sense transcription would be white (more clearly unchanging) when the scale is adjusted.

We agree and have done this. The reviewer is correct that most sense transcription is unchanged by INTS11 loss.  However, as we alluded to in the original submission, a subset of transcripts shows a promoter-proximal increase after INTS11 depletion. We have expanded the analyses of this effect (see responses to other comments) but stress that it is neither as general nor as large as the antisense effect.

The authors make the point that there is mildly increased transcription over the 5' end of some genes upon INST11 depletion and show a track (Supplementary Fig 2A). It is not immediately obvious from the presentation of the meta-analysis in Figure 2D how generalisable this statement is. Perhaps the size of the panel or thickness of the lines in Figure 2D could be adjusted so that the peak of the control (in blue) could be seen. Perhaps an arrow indicating the peak could be added? I'm assuming the peak at the TSS is slightly lower in the control compared to INST11 depletion based on the authors' statement.

We have provided multiple new analyses of this data to highlight where there are promoter-proximal effects of INTS11 loss in the sense direction.  Please see our response to the public review of reviewer 1 and new supplemental figures 2A, 2B, 4A and 4B which highlight the sense transcription increased in the absence of INTS11.

The authors label Figure 4 "Promoters lose their directionality when CDK9 is inhibited" - but in INST11 depleted cells treated with CDK9i they find that there still is a bias towards sense transcription. Suggested edit "Some promoter directionality is lost when CDK9 is inhibited" or similar.

We agree and have made this change.

The authors conclude that INTS11-mediated effects are the result of perturbation of the catalytic activities of Integrator, the authors should perform rescue experiments with the catalytically dead E203Q-INTS11 mutant.

This is a very good suggestion and something we had intended to pursue.  However, as we will describe below (and shown in Supplemental Figure 4G), there were confounding issues with this experiment.

The E203Q mutant of INTS11 is widely used in the literature to test for catalytic functions of INTS11.  However, we have found that this mutation impairs the ability of INTS11 to bind other Integrator modules in cells. Based on co-immunoprecipitation of flag-tagged WT and E203Q derivatives, INTS1 (backbone module), 10 (tail module), and 8 (phosphatase module) all show reduced binding to E203Q vs. WT. Because E203Q INTS11 is defective in forming Integrator complexes, rescue experiments might not fully distinguish the effects of INTS11 activity from those caused by defects in complex assembly. While this may at first seem unexpected, in the analogous 3’ end processing complex, catalytic mutants of CPSF73 (which is highly related to INTS11) negatively affect its interaction with other complex members (Kolev and Steitz, EMBO Reports 2005).

We hypothesise that INTS11 activity is most likely involved in attenuating promoter-proximal transcription, but we cannot formally rule out other explanations and discuss this in our revision. Regardless of how INTS11 attenuates transcription, our main conclusion is on its requirement to terminate antisense transcription whether this involves its cleavage activity or not.

The authors suggest that CDK9 modulates INTS11 activity/assembly and suggest this may be related to SPT5. Is there an effect of CDK9 inhibition on the snRNA's highlighted in Figure 2B?

We believe that snRNAs are different from protein-coding genes concerning CDK9 function. Shona Murphy’s lab previously showed that, unlike protein-coding genes, snRNA transcription is insensitive to CDK9 inhibition, and that snRNA processing is impaired by CDK9 inhibition (Medlin et al., EMBO 2003 and EMBO 2005).  We reproduce these findings by metaanalysis of 15 highly expressed and well-separated snRNAs and by qRT-PCR of unprocessed RNU1-1, RNU5A-1 and RNU7-1 snRNA following CDK9 inhibition. We observe snRNA read-through by POINT-seq following INTS11 loss whether CDK9 is inhibited or not (left panel, below). Note the higher TES proximal signal in CDK9i conditions, which likely reflects the accumulation of unprocessed snRNA as validated by qPCR for three example snRNAs (right panel, below).

Author response image 1.

For Figure 4, would similar results be observed using inhibitors targeting other transcriptional CDKs such as CDK7,12/13?

In response to this suggestion, we analysed four selected protein-coding transcripts (the same 4 that we used to validate the CDK9i results) by qRT-PCR in a background of CDK7 inhibition using the THZ2 compound (new Supplemental Figure 4E).  THZ2 suppresses transcription from these genes as expected.  Interestingly, expression is restored by co-depleting Integrator, recapitulating our findings with CDK9 inhibition.  As CDK7 is the CDK-activating kinase for CDK9, its inhibition will also inhibit CDK9 so THZ2 may simply hit this pathway upstream of where CDK9 inhibitors.  Second, CDK7 may independently shield transcription from INTS11.  We allude to both interesting possibilities.

What happens to the phosphorylation state of anti-sense engaged RNAPII when INTS11 is acutely depleted and/or CDK9 is inhibited? This could be measured by including Ser5 and Ser2 antibodies in the sPOINT-seq assay and complemented with Western Blot analysis.

We have performed the western blot for Ser5 and Ser2 phosphorylation as suggested.  Both signals are mildly enhanced by INTS11 loss, which is consistent with generally increased transcription.  Ser2p is strongly reduced by CDK9 inhibition, which is consistent with the loss of nascent transcription in this condition.  Interestingly, both modifications are partly recovered when INTS11 is depleted in conjunction with CDK9 inhibition. This is consistent with the effects that we see on POINT-seq and shows that the recovered transcription is associated with some phosphorylation of RNAPII CTD.  This presumably reflects the action(s) of kinases that can act redundantly with CDK9.

We have not performed POINT-seq with Ser5p and Ser2p antibodies under these various conditions.  Our rationale is that our existing data uses an antibody that captures all RNAPII (regardless of its phosphorylation status), which we feel most comprehensively assays transcription in either direction. Moreover, the lab of Fei Chen (Hu et al., Mol Cell 2023) recently published Ser5p and Ser2p ChIP-seq following INTS11 loss. By ChIP-seq, they observe a bigger increase in antisense RNAPII occupancy vs. sense providing independent and orthogonal support for our POINT-seq data.  Interestingly, this antisense increase is not paralleled by proportional increases in Ser5p or Ser2p signals.  This suggests that the unattenuated antisense transcription resulting from INTS11 loss does not have high Ser5p or Ser2p.  Since CDK7 and 9 are major Ser5 and 2 kinases, this supports our model that their activity is less prevalent for antisense transcription.  We now discuss these data in our revision.   

The HIV reporter RNA experiments should be performed with the CDK9 inhibitor added to the experimental conditions. Presumably CDK9 inhibition would result in no upregulation of the reporter upon addition of TAT and/or dTAG. Perhaps the amount of TAT should be reduced to still have a dynamic window in which changes can be detected. It is possible that reporter activation is simply at a maximum. Can anti-sense transcription be measured from the reporter?

We have performed the requested CDK9 inhibitor experiment to confirm that TAT-activated transcription from the HIV promoter is CDK9-dependent (new supplemental figure 4F).  Consistent with previous literature on HIV transcription, CDK9 inhibition attenuates TAT-activated transcription.  Importantly, and in line with our other experiments, depletion of INTS11 results in significant restoration of transcription from the HIV promoter when CDK9 is inhibited. Thus, TAT-activated transcription is CDK9-dependent and, as for endogenous genes, CDK9 prevents attenuation by INTS11.

While TAT-activated transcription is high, we do not think that the plasmid is saturated. When considering this question, we revisited previous experiments using this system to study RNA processing (Dye et al., Mol Cell 1999, Cell 2001, Mol Cell 2006). In these cases, mutations in splice sites or polyadenylation sites have a strong effect on RNA processing and transcription around HIV reporter plasmids. Effects on transcription and RNA processing are; therefore, apparent in the appropriate context. In contrast, we find that the complete elimination of INTS11 has no impact on RNA output from the HIV reporter. Our original experiment assessing the impact of INTS11 loss in +TAT conditions used total RNA.  One possibility is that this allows non-nascent RNA to accumulate which might confound our interpretation of INTS11 effects on ongoing transcription.  However, the new experiment described in the paragraph above was performed on chromatin-associated (nascent) RNA to rule this out.  This again shows no impact of INTS11 loss on HIV promoter-derived transcription in the presence of TAT.

To our knowledge, antisense transcription is not routinely assayed from plasmids. They generally employ very strong promoters (e.g. CMV, HIV) to drive sense transcription.  Crucially, their circular nature means that RNAPII going around the plasmid could interfere with antisense transcription coming the other way which does not happen in a linear genomic context. This is why we restricted our use of plasmids to looking at the effects of stimulated CDK9 recruitment (via TAT) on transcription rather than promoter directionality.   

The authors should clearly state how many replicates were performed for the genomics experiments. Ideally, a signal should be quantified and compared statistically rather than relying on average profiles only.

We have stated the replicate numbers for sequencing experiments in the relevant figure legends. All sequencing experiments were performed in at least two biological replicates, but often three. In addition, we validated their key conclusions by qPCR or with orthogonal sequencing approaches.

Reviewer #3 (Recommendations For The Authors):

The authors provide strong evidence in support of their claims.

ChIP-seq of pol2S5 and S2 upon INST11 and CDK9 inhibition will strengthen the observation that transcription in the sense direction is more efficient.

We view the analysis of total RNAPII as the most unbiased way of establishing how much RNAPII is going one way or the other. Importantly, ChIP-seq was very recently performed for Ser2p and Ser5p RNAPII derivatives in the lab of Fei Chen (Hu et al., Mol Cell 2023). Their data shows that loss of INTS11 increases the occupancy of total RNAPII in the antisense direction more than in the sense direction, which is consistent with our finding. Interestingly, the increased antisense RNAPII was not paralleled with an increase in Ser2p or Ser5p. This suggests that, following INTS11 loss, the unattenuated antisense transcription is not associated with full/normal Ser2p or Ser5p. These modifications are normally established by CDK7 and 9; therefore, this published ChIP-seq suggests that they are not fully active on antisense transcription when INTS11 is lost. This supports our overall model that CDK9 (and potentially CDK7 as suggested for a small number of genes in new Supplemental Figure 4E) is more active in the sense direction to prevent INTS11-dependent attenuation. We now discuss these data in our revision.

In Supplementary Figure 2, the eRNA expression increases upon INST11 degradation, I wonder if the effects of this will be appreciated on cognate promoters? Can the authors test some enhancer:promoter pairs?

We noticed that some genes (e.g. MYC) that are regulated by enhancers show reduced transcription in the absence of INTS11. Whilst this could suggest a correlation, the transcription of other genes (e.g. ACTB and GAPDH) is also reduced by INTS11 loss although they are not regulated by enhancers.  A detailed and extensive analysis would be required to establish any link between INTS11-regulated enhancer transcription and the transcription of genes from their cognate promoters.  We agree that this would be interesting, but it seems beyond the scope of our short report on promoter directionality.

Line 111, meta plot was done of 1316 genes. Details on this number should be provided. Overall, the details of methods and analysis need improvement. The layout of panels and labelling on graphs can be improved.

We have now explained the 1316 gene set.  In essence, these are the genes separated from an expressed neighbour by at least 10kb.  This distance was selected because depletion of RBBP6 induces extensive read-through transcription beyond the polyadenylation site of protein-coding genes.  To avoid including genes affected by transcriptional read-through from nearby transcription units we selected those with a 10kb gap between them. This was the only selection criteria so is unlikely to induce any unintended biases. Finally, we have added more information to the figure panels and their legends, which we hope will make our manuscript more accessible.

eLife assessment

The important study uses a new experimental method to provide compelling evidence on how sense- and anti-sense transcription is differentially regulated. The method described here can generally be used to study the alterations in transcription. This paper will be of interest to scientists working in the gene regulation community.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Eaton et al. examine the regulation of transcription directionality using a powerful genomic approach (more about the methodology below).<br /> Their data challenge the notion that the polyadenylation signal-reading Cleavage and Polyadenylation (CPA) complex is responsible for controlling promoter directionality by terminating antisense transcription. Namely, depletion of the required CPA factor RBBP6 has little effect on antisense transcription measured by POINT. They find instead that initiation is intrinsically preferential in the sense direction and additionally maintained by the activities of an alternative processing complex called Integrator, together with the kinase CDK9. In the presence of CDK9 activity, depletion of Integrator endoribonuclease INTS11 leads to globally increased transcription in the antisense direction, and minor effects in the sense direction. However, CDK9 inhibition reveals that sense transcription is also sensitive to INS11 depletion. The authors suggest that CDK9 activity is stronger in the sense direction, preventing INTS11-mediated premature termination of sense transcripts.

Strengths:

The combination of acute depletion of the studied factors using degron approaches (important to limit possible secondary effects), together with novel and very sensitive nascent transcriptomics methods POINT and sPOINT is very powerful. The applied spike-in normalization means the analysis is more rigorous than most. Using this methodology allowed the authors to revisit the interesting question of how promoter/transcription directionality is determined.

The data quality appears very good and the fact that both global analysis as well as numerous gene-specific examples are shown makes it convincing.

The manuscript is well written and hence a pleasure to read.

Weaknesses:

The bias in transcriptional initiation directionality remains to be elucidated.

Conclusion/assessment:

This important work substantially advances our understanding of the mechanisms governing the directionality of human promoters. The evidence supporting the claims of the authors is compelling, with a.o. the use of advanced nascent transcriptomics including spike-in normalization controls and acute protein depletion using degron approaches.

In my opinion the authors' conclusions are well supported.

Not only the manuscript but also the data generated will be useful to the wide community of researchers studying transcriptional regulation. Also, the POINT-derived novel sPOINT method described here is very valuable and can positively impact work in the field.

Reviewer #2 (Public Review):

Summary:

Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper which uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:

One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

Weaknesses:

While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figure 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

Reviewer #3 (Public Review):

Summary:

Using protein degradation approach, Eaton et al show that INST11 can terminate the sense and anti-sense transcription but higher activity of CDK9 in sense direction protects it from INS11-dependent termination. They developed sPOINT-seq that detects nascent 5'-capped RNA. The technique allowed them to reveal robust transcription initiation of sense-RNA as compared to anti-sense.

Strengths:

The strength of paper is acute degradation of proteins, eliminating the off-target effects. Further, the paper uses elegant approaches such as POINT and sPOINT-seq to measure nascent RNA and 5'-capped short RNA. Together, the combination of these three allowed the authors to make clean interpretations of data.

Weaknesses:

While manuscript is well written, the details on panel is not sufficient. The methods can be more elaborate for better understanding. Additional discussion on how authors findings contradict the existing model of anti-sense transcription termination should be added.

in the revised manuscript, authors have added details on panels and elaborated method and other sections for better understanding.

トル

eLife assessment

The study presents valuable findings on the molecular mechanisms of glucose-stimulated insulin secretion from pancreatic islets, focusing on the main regulatory elements of the signaling pathway in physiological conditions. While the evidence supporting the conclusions is solid, the study can be strengthened by the use of a beta cell line or knockout mice. The work will be of interest to cell biologists and biochemists working on diabetes.

Reviewer #1 (Public Review):

Summary:

This study investigated the mechanism by which PGE2 inhibits the release of insulin from pancreatic beta cells in response to glucose. The researchers used a combination of cell line experiments and studies in mice with genetic ablation of the Kv2.2 channel. Their findings suggest a novel pathway where PGE2 acts through EP2/EP4 receptors to activate PKA, which directly phosphorylates a specific site (S448) on the Kv2.2 channel, inhibiting its activity and reducing GSIS.

Strengths:

- The study elegantly demonstrates a potential pathway connecting PGE2, EP2/EP4 receptors, PKA, and Kv2.2 channel activity, using embryonic cell line.<br /> - Additional experiments in INS1 and primary mouse beta cells with altered Kv2.2 function partially support the inhibitory role of PGE2 on GSIS through Kv2.2 inhibition.

Weaknesses:

- A critical limitation is the use of HEK293T cells, which are not pancreatic beta cells. Functional aspects can differ significantly between these cell types.<br /> - The study needs to address the apparent contradiction of PKA activating insulin secretion in beta cells, while also inhibiting GSIS through the proposed mechanism.<br /> - A more thorough explanation is needed for the discrepancies observed between the effects of PGE2 versus Kv2.2 knockdown/mutation on the electrical activity of beta cells and GSIS.

Reviewer #2 (Public Review):

The authors identified new target elements for prostaglandin E2 (PGE2) through which insulin release can be regulated in pancreatic beta cells under physiological conditions. In vitro extracellular exposure to PGE2 could directly and dose-dependently inhibit the potassium channel Kv2.2. In vitro pharmacology revealed that this inhibition occurs through the EP2/4 receptors, which activate protein kinase A (PKA). By screening specific sites of the Kv2.2 channel, the target phosphorylation site (S448) for PKA regulation was found. The physiological relevance of the described signaling cascade was investigated and confirmed in vivo, using a Kv2.2 knockdown mouse model.

The strength of this manuscript is the novelty of the (EP2/4-PKA-Kv2.2 channel) molecular pathway described and the comprehensive methodological toolkit the authors have relied upon.

The introduction is detailed and contains all the information necessary to place the claims in context. Although the dataset is comprehensive and a logical lead is consistently built, there is one important point to consider: to clarify that the described signaling pathway is characteristic of normal physiological conditions and thus differs from pathological changes. It would be useful to carry out basic experiments in a diabetes model (regardless of whether this is in mice or rats).

Author response:

We thank the reviewers for their positive evaluation and constructive feedback on our study.

We acknowledge the concern regarding the use of HEK293T cells. In the revised manuscript, we will provide a more detailed explanation of the role of the PKA pathway in the regulation of GSIS by PGE2. To validate this regulation through Kv2.2, we will overexpress the Kv2.2 mutant channel in beta cells and assess its impact. Additionally, we will verify the specificity of the antibodies for EP1-EP4 receptors by knockdown. To confirm the receptors involved in PGE2 function, we will use additional EP receptor blockers or perform receptor knockdown experiments.

We will clarify that the described signaling pathway operates under normal physiological conditions and differs from pathological changes.

We once again thank the reviewers for their positive evaluation and constructive suggestions.

eLife assessment

This work describes a novel affinity interactomics approach that allows investigators to identify networks of protein-protein interactions in cells. The important findings presented here describe the application of this technique to the SH3 domain of the membrane remodeling Bridging Integrator 1 (BIN1), the truncation of which leads to centronuclear myopathy. The authors present solid evidence that BIN1 SH3 engages with an unexpectedly high number of cellular proteins, many of which are linked to skeletal muscle disease, and evidence is presented to suggest that BIN1 may play a role in mitosis creating the potential for new avenues in drug development efforts. Some of the findings, however, remain rather preliminary, lack sufficient replicates and may require additional experiments to definitively support the conclusions.

Reviewer #1 (Public Review):

Summary:

In this paper, Zambo and coworkers use a powerful technique, called native holdup, to measure the affinity of the SH3 domain of BIN1 for cellular partners. Using this assay, they combine data using cellular proteins and proline-containing fragments in these proteins to identify 97 distinct direct binding partners of BIN1. They also compare the binding interactome of the BIN1 SH3 domain to the interactome of several other SH3 domains, showing varying levels of promiscuity among SH3 domains. The authors then use pathway analysis of BIN1 binding partners to show that BIN1 may be involved in mitosis. Finally, the authors examine the impact of clinically relevant mutations of the BIN1 SH3 domain on the cellular interactome. The authors were able to compare the interactome of several different SH3 domains and provide novel insight into the cellular function of BIN1. Generally, the data supports the conclusions, although the reliance on one technique and the low number of replicates in each experiment is a weakness of the study.

Strengths:

The major strength of this paper is the use of holdup and native holdup assays to measure the affinity of SH3 domains to cellular partners. The use of both assays using cell-derived proteins and peptides derived from identified binding partners allows the authors to better identify direct binding partners. This assay has some complexity but does hold the possibility of being used to measure the affinity of the cellular interactome of other proteins and protein domains. Beyond the utility of the technique, this study also provides significant insight into the cellular function of BIN1. The authors have strong evidence that BIN1 might have an undiscovered function in cellular mitosis, which potentially highlights BIN1 as a drug target. Finally, the study provides outstanding data on the cellular binding properties and partners of seven distinct SH3 domains, showing surprising differences in the promiscuity of these proteins.

Weaknesses:

There are several weaknesses of the study. First, the authors rely completely on a single technique to measure the affinity of the cellular interactome. The native holdup is a relatively new technique that is powerful yet relatively unproven. However, it appears to have the capacity to measure the relative affinity of proteins and the authors describe the usefulness of the technique. Second, and most important, the authors use a relatively small number of replicates for the holdup assays. The holdup technique will have biological variation in the cellular lysate or purified protein that could impact the results, so more replicates would enhance the reliability of the results.

Reviewer #2 (Public Review):

Summary:

The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

Strengths:

The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain.

Weaknesses:

Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

Author response:

The following is the authors’ response to the previous reviews.

Reviewer #1

We modified the text regarding PRC1 according to the reviewer’s recommendation.

Reviewer #2

Following the reveiwer’s advise, we introduced the holdup assay, as well as the native holdup assay in more details.

This new part now also discusses the question of replicates in more details. We do not agree with the eLife assessment on this matter, but we think that this assessment was made because analyzing holdup data requires a different approach compared to more conventional interactomic approaches and these differences were not introduced in sufficient depth. We hope that the inclusion of more background reasoning, as well as by providing a more detailed comparison of the measured independent BIN1 interactomes, now included on Figure S4, will eliminate all confusion in the reader.

We thank the reviewer for guiding us to a previous work that was done on Grb2. Indeed, the finding of this earlier work aligns perfectly with our finding suggesting general similarities in SH3 domain mediated interactions.

Underlying issues (underlying crisis) need to transform into a thorn in the side of the government in order to create an existential crisis for the government in order to be taken seriously

Governments need to be forced to do something that's radical - they prefer keeping things the same. Why? Because doing more is consumption of more energy, it's politically dangerous, doing anything out of ordinary raises accountability.

Résumé de la vidéo [00:00:00][^1^][1] - [00:20:41][^2^][2]:

Cette vidéo présente une discussion approfondie sur l'éducation, en se concentrant sur les idées fausses courantes concernant le système scolaire et l'impact des stéréotypes de genre. Louise Tourret, journaliste et productrice de l'émission "Être et savoir" sur France Culture, partage ses observations et expériences pour démystifier ces idées reçues et souligner l'importance d'une approche réfléchie de l'éducation.

Points forts: + [00:00:13][^3^][3] Introduction de Louise Tourret * Présentation de son ouvrage sur les idées fausses de l'école * Expérience en tant que journaliste et productrice * Brève expérience en tant qu'enseignante contractuelle + [00:02:08][^4^][4] Impact des stéréotypes de genre dans l'éducation * Influence des stéréotypes sur les élèves et les enseignants * Réflexion sur la mixité et l'égalité de genre à l'école * Importance de repenser les pratiques éducatives + [00:11:02][^5^][5] Déconstruction des mythes éducatifs * Discussion sur l'école et le sexisme * Examen des idées reçues sur les élèves à haut potentiel intellectuel (HPI) * Importance de valoriser l'expérience des enseignants + [00:16:17][^6^][6] Rôle de la formation continue des enseignants * Interrogation sur le développement professionnel des enseignants * Impact des idées reçues sur les pratiques pédagogiques * Nécessité d'une réflexion collective sur l'éthique de l'enseignement

Résumé de la vidéo [00:20:43][^1^][1] - [00:41:03][^2^][2] : La vidéo aborde la question de l'éducation en France, en examinant les défis et les perspectives d'avenir du système éducatif. Elle soulève des préoccupations sur la manière dont l'école peut créer une distance sociale plutôt que de rapprocher les gens, et comment cela peut influencer les attitudes politiques et sociales. La vidéo explore également les différences entre les pédagogies françaises et nordiques, l'importance de la culture générale dans l'éducation française, et la nécessité d'adapter l'école aux besoins individuels des élèves tout en préservant l'héritage culturel.

Points saillants: + [00:20:43][^3^][3] Les défis de l'éducation en France * Discussion sur l'impact de l'éducation sur les choix politiques * Analyse du ressentiment développé à l'école envers les diplômés * Réflexion sur l'humiliation et le sentiment de perte dans le système scolaire + [00:23:00][^4^][4] Comparaison avec les pédagogies nordiques * Évaluation des idées fausses sur l'adoption des pédagogies nordiques * Importance des maîtres spécialisés et de la formation continue en Finlande * Différences culturelles dans la perception de l'enfant et de l'élève + [00:26:01][^5^][5] La culture générale et l'éducation * La valeur de la culture générale dans l'éducation française * Transmission du patrimoine culturel et révérence pour celui-ci * Tension entre l'héritage culturel et un projet d'école pour tous + [00:31:01][^6^][6] L'évaluation et la sélection dans l'éducation * Débat sur l'efficacité de l'évaluation de 0 à 20 * Impact de Parcoursup et de la sélection basée sur les notes * Importance de diversifier les notions de réussite scolaire + [00:34:33][^7^][7] La forme scolaire et les méthodes d'enseignement * Discussion sur l'enseignement mutuel et les alternatives à la forme scolaire traditionnelle * Réflexion sur la liberté pédagogique et l'auto-évaluation des enseignants * L'importance de l'adaptation des espaces éducatifs et de la coopération

Résumé de la vidéo [00:41:06][^1^][1] - [00:55:48][^2^][2]:

Cette partie de la vidéo aborde les stéréotypes de genre dans l'éducation, la réussite scolaire des filles par rapport aux garçons, et les défis rencontrés par les filles dans les domaines des mathématiques et des sciences. L'intervenant discute également de l'impact des neurosciences sur l'éducation, les mythes qui les entourent, et la manière dont les enseignants peuvent intégrer ces connaissances dans leur pratique pédagogique.

Points forts: + [00:41:06][^3^][3] Stéréotypes de genre et éducation * Discussion sur la réussite des filles à l'école * Les garçons s'orientent vers des filières plus prestigieuses * Questionnement sur la représentation des femmes dans les sciences + [00:44:01][^4^][4] Impact des neurosciences sur l'éducation * Les neurosciences apportent des connaissances mais pas de solutions toutes faites * Prudence face aux neuromythes et à l'application pratique * Importance de la formation des enseignants et de l'adaptation pédagogique + [00:49:01][^5^][5] La coopération dans l'éducation * La coopération est essentielle mais souvent limitée par le système * Les contradictions dans les pratiques éducatives * L'importance de la réflexion sur la pratique professionnelle